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a r t i c l e i n f o a b s t r a c t
Article history: Translation is one of the fundamental processes of life. It comprises the assembly of polypeptides whose
Received 20 March 2017 amino acid sequence corresponds to the codon sequence of an mRNAs ORF. Translation is performed by
Received in revised form 25 April 2017 the ribosome; therefore, in order to understand translation and its regulation we must be able to deter-
Accepted 29 May 2017
mine the numbers and locations of ribosomes on mRNAs in vivo. Furthermore, we must be able to exam-
Available online xxxx
ine their redistribution in different physiological contexts and in response to experimental
manipulations. The ribosome profiling method provides us with an opportunity to learn these locations,
Keywords:
by sequencing a cDNA library derived from the short fragments of mRNA covered by the ribosome. Since
RNA
Translation
its original description, the ribosome profiling method has undergone continuing development; in this
Ribosome article we describe the methods current state. Important improvements include: the incorporation of
Ribosome profiling sample barcodes to enable library multiplexing, the incorporation of unique molecular identifiers to
High-throughput sequencing enable to removal of duplicated sequences, and the replacement of a gel-purification step with the enzy-
RNA-sequencing matic degradation of unligated linker.
2017 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://
creativecommons.org/licenses/by/4.0/).
http://dx.doi.org/10.1016/j.ymeth.2017.05.028
1046-2023/ 2017 The Authors. Published by Elsevier Inc.
This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Please cite this article in press as: N.J. McGlincy, N.T. Ingolia, Methods (2017), http://dx.doi.org/10.1016/j.ymeth.2017.05.028
2 N.J. McGlincy, N.T. Ingolia / Methods xxx (2017) xxxxxx
metazoans. For recent perspectives on ribosome profiling and its This RNA-DNA hybrid molecule is used as a template for reverse
applications, readers are referred to several excellent reviews: transcription, and following gel-based purification of full-length
Jackson & Standart [6], Ingolia [7], Brar & Weissman [8], and products the resulting single-stranded cDNA is then circularized
Andreev et al. [9]. using a CircLigase. A double-stranded DNA library of suitable
This article describes the current state of the ribosome profiling structure and concentration for Illumina sequencing is then con-
protocol as routinely performed in our laboratory. It has been the structed from the single-stranded cDNA circles by means of a
basis of ribosome profiling experiments from recent studies associ- PCR reaction.
ated with our laboratory, including but not limited to: Iwasaki et al. As the result of our continuing development and optimization,
[10], Werner et al. [11], and Ishikawa et al. [12]. In essence, the pro- the protocol described in this article has a number of innovations
cedure is similar to that described in Ingolia et al. [13]: cells are with respect to our previously published protocol (see Fig. 1):
rapidly harvested and lysed under conditions that are expected
to preserve in vivo ribosome positioning. This cell lysate is then 1. We have designed a number of oligonucleotide synthetic con-
subjected to nuclease footprinting with RNase I. Ribosomes are trols to guide the remaining gel extraction steps, judge the effi-
pelleted from the digested lysate by ultracentrifugation through cacy of the circularization reaction, and to aid in the planning
a sucrose cushion, and then RNA is extracted from the pellet. To and execution of the final library construction PCR. These are
isolate ribosome footprints, the RNA from the ribosomal pellet is outlined in Section 2.3.
resolved by electrophoresis through a denaturing gel, and then 2. A number of the enzymes employed in the footprinting and
fragments of the expected size range extracted from the gel. The library construction have changed: we now obtain RNase I from
0
3 ends of these RNA fragments are treated with T4 PNK to allow Epicentre; use Protoscript II for the reverse transcription, and
ligation of a pre-adenylated DNA linker with T4 Rnl2(tr) K227Q. CircLigase II for cDNA circularization.
Fig. 1. A schematic of the ribosome profiling protocol. The major steps of the updated ribosome profiling protocol, highlighting several of the most important developments.
Each step has the same name and number as the corresponding part of this protocol. UMI Unique Molecular Index.
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Table 1
Equipment and reagents used in ribosome profiling.
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4 N.J. McGlincy, N.T. Ingolia / Methods xxx (2017) xxxxxx
Table 1 (continued)
1. Cycloheximide is very toxic and harmful to the environment. Handle solutions containing cycloheximide with care, and decontaminate and dispose of waste in accordance
with institutional regulations.
2. CAUTION Harringtonine is very toxic. Handle solutions containing harringtonine with care, and decontaminate and dispose of waste in accordance with institutional
regulations.
3. CAUTION Liquid nitrogen is a refrigerated liquid. It may cause burns or injury, and may displace oxygen and thus cause rapid suffocation.
4. Epicentre and Life Technologies, (the two commercial suppliers of Escherichia coli RNase I) use different unit definitions for RNase I. Substantial changes in the RNase
activity during nuclease footprinting could compromise the experiment.
5. Can be substituted with other Direct-zol compatible reagents. CAUTIONTRIzol and equivalent reagents are very toxic, corrosive, and volatile. Use proper protection when
using TRIzol, perform work in a fume hood, and dispose all waste in accordance with institutional regulations.
6. CAUTION Highly flammable and volatile.
7. CAUTION Irritant.
8. CAUTION Reproductive toxin.
9. CAUTION Acrylamide is a neurotoxin. Use proper protection when handling polyacrylamide gels.
10. CAUTION Nucleic acid stains are typically mutagenic. Use personal protection when handling gel staining solution and dispose of waste in accordance with regulations.
0
11. Contains Mth RNA Ligase, supplied with 1 mM ATP and 10X 5 DNA adenylation reaction buffer.
0
12. Supplied with 10X T4 polynucleotide kinase buffer. CRITICAL - Avoid the 3 phosphatase minus mutant, M0236S.
13. Supplied with PEG 8000 50% w/v and 10X T4 RNA ligase buffer.
14. CRITICAL The New England Biolabs unit definition for RecJ is 1/20th the Epicentre unit definition. CRITICAL Epicentre now recommends that RecJ is stored at 80 C to
preserve the enzymes activity.
15. Supplied with 5X first-strand buffer and 0.1 M DTT. NOTE Similar reverse transcriptases such as SuperScript II (Invitrogen) can be substituted directly; SuperScript III
can be used with minor alterations in the reverse transcription protocol.
16. CAUTION Highly corrosive.
17. Supplied with 10X CircLigase buffer, 5 M Betaine, and 50 mM MnCl2.
18. Supplied with 5X HF buffer.
19. A standard UV transilluminator can be used instead.
0
3. We have found that a shorter, faster ultracentrifugation step is 5 -deadenylase is used to deadenylate the pre-adenylated lin-
equally effective in pelleting the ribosome. For such a long, lin- ker, rendering it the only molecular species in the ligation vul-
0 0
ear protocol time savings are a significant advantage. We now nerable to degradation by the 5 -3 ssDNA exonuclease RecJ. As
recommend centrifugation at 100 krpm for 1 h at 4 C in a gel extractions typically result in a loss of material, we expect
TLA100.3 or TLA110 rotor (see Section 3.2). this replacement will make the protocol more efficient.
4. For a number of steps in the protocol, we now employ specific 7. We now perform rRNA reduction on linker-ligated RNA frag-
kits. For the purification of RNA from the ribosome pellet we ments prior to reverse transcription using the Ribo-Zero Gold
use the Direct-zol kit from Zymo Research (see Table 1). Addi- rRNA removal kits from Illumina (see Table 1).
tionally, for a number of buffer exchanges we now utilize the 8. In contrast to the library construction PCR optimization per-
(Oligo) Clean & Concentrator kit, also from Zymo Research formed in Ingolia et al. [13], we have implemented a qPCR assay
(see Table 1). The use of these kits has improved the repro- to assess the concentration of the single-stranded cDNA circles
ducibility and convenience of the corresponding steps in the produced by the circularization reaction. This is used to deter-
protocol. mine the optimal conditions (amount of circularized template
5. We have implemented a series of custom DNA linkers with and number of cycles) for a single library construction PCR. This
sequences that allow for the identification of individual samples procedure has the added benefit of preserving more cDNA cir-
(sample barcodes) and individual ligation events (unique cles for future use.
molecular identifiers, UMIs). This strategy provides a number
of advantages: first, sample barcodes enable sample multiplex- To illustrate these advances this article follows the production
ing before sequencing. This allows more samples to be of a ribosome profiling library consisting of five distinct wild-
sequenced for the same cost. Furthermore, it can be used to type yeast samples. We also discuss data analysis, and use the con-
avoid the variance that is associated with sequencing different structed library to illustrate the characteristics we expect of ribo-
samples on different lanes of a sequencer [14]. Secondly, the some footprints. We then conclude by discussing the variety of
UMIs are likely to ameliorate the effect of sequence biases in ribosome profiling protocols that exist, and the experimental
ligation and circularization during library construction choices they present.
[15,16]. Thirdly, the UMIs will also allow for the computational
removal of sequences likely to derive from selective amplifica-
tion during the library construction PCR [15,16]. 2. Materials & methods
6. Ingolia et al. [13] includes a gel extraction to separate ligated
RNA fragments from unligated DNA linker. We have replaced Similarly to other targeted high-throughput sequencing meth-
this step with an enzymatic depletion of unligated linker. Yeast ods, ribosome profiling requires a large variety of equipment and
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N.J. McGlincy, N.T. Ingolia / Methods xxx (2017) xxxxxx 5
reagents. In Table 1 we detail the equipment and reagents we rou- 2.1.6. DNA gel extraction buffer
tinely use to perform ribosome profiling. In Section 2.1 we describe See Table 5. Prepare in advance and store indefinitely at room
the recipes of the buffers we employ. In Section 2.2 we detail the temperature.
sequences of the oligonucleotides we use in preparation of the
ribosome profiling sequencing library, and in Section 2.3 the syn- 2.1.7. 6X Non-denaturing sample loading buffer
thetic controls we use to monitor our progress throughout the See Table 6. Prepare in advance and store at room temperature.
library preparation procedure. Other standard DNA non-denaturing electrophoresis sample load-
ing buffers can be substituted.
2.1. Buffer recipes
2.2. Oligonucleotides
Use sterile, RNase-free water, reagents, and techniques in all
recipes and protocol steps. Oligonucleotide sequences are described using the standard
nomenclature, outlined in Table 7.
Code Meaning
Table 4 [ACGT] Nucleotides
RNA gel extraction buffer recipe. N A random nucleotide
r[ACGUN] Ribonucleotides
Component Final concentration 0
/5Phos/ 5 phosphorylation
0
NaOAc pH 5.5 300 mM /3Phos/ 3 phosphorylation
EDTA 1 mM /iSp18/ An 18-atom hexa-ethyleneglycol spacer
SDS 0.25% v/v /3ddC/ Dideoxycytidine
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6 N.J. McGlincy, N.T. Ingolia / Methods xxx (2017) xxxxxx
Table 8
Linker oligonucleotide sequences.
multiplexing of samples before the reverse transcription step 2.2.4. qPCR quantitation primers
(Section 3.6). Each sample barcode differs from each other sample Forward qPCR primer NI-827:
0
barcode at three or more nucleotide positions. Moreover, the 5 -CTCTTTCCCTACACGACGCTC
nucleotide composition of each sample barcode is roughly bal- Reverse qPCR primer NI-828:
0
anced and is interleaved with consideration of purines and pyrim- 5 -GTGACTGGAGTTCAGACGTGTG
idines, A/T versus G/C base pairs, and distinct excitation during
Illumina sequencing (AC versus GT). The sequences of these linkers
are enumerated in Table 8.
2.2.5. Library construction PCR primers
Forward library PCR primer, NI-NI-798:
0
5 -AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACG
2.2.2.1. Enzymatic pre-adenylation of linker using Mth RNA ligase.
CTC
Linker oligonucleotides require enzymatic pre-adenylation prior to
In addition to barcodes added during linker ligation, different
ligation to RNA fragments with T4 Rnl2(tr) K227Q.
pools can be barcoded at the PCR stage using indexed reverse
library PCR primers the same approach as the TruSeq low-
1. Combine 1.2 ll linker oligonucleotide at 100 lM, 2 ll 10X 5
0
Table 9
Indexed reverse library PCR primers.
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2.3.1. Post-ligation, NI-805, Index: CGATC 7. Cap the tube of yeast lysate in liquid nitrogen using a pierced
0
5 -rArUrGrUrUrArGrGrGrArUrArArCrArGrGrGrUrArAr cap and place it upright in a 80 C freezer until the liquid
UrGrCrGrANNNNNCGATCTGATCGGAAGAGCAC ACGTCTGAArC nitrogen evaporates. Frozen yeast lysate can be stored indefi-
nitely at 80 C.
2.3.2. RT Product, NI-804, Index: GACTG 8. Thaw the yeast lysate gently and immediately centrifuge the
0
5 -/5Phos/NNAGATCGGAAGAGCGTCGTGTAGGGAAAGAG/iSp18/ tube at 3000g, 4 C for 5 min and recover the supernatant into
GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCACAGTCNNNNNTCGCAT one or more non-stick RNase-free microfuge tubes. Further clar-
TACCCTGTTATCCCTAACAT ify the supernatant by spinning at 20,000g, 4 C for 10 min and
recover the supernatant, typically 1.5 ml.
2.3.3. Circularization Product, NI-803, Index: CATGC 9. Take 50 ll aliquots of this yeast lysate and flash freeze by
0
5 -GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCAGCATGNNNNNT immersion in liquid nitrogen. This lysate can be stored indefi-
CGCATTACCCTGTTATCCCTAACATNNAGATCGGAAGAGCGTCGTGTAGG nitely at 80 C. Yeast lysate prepared in this fashion is typi-
GAAAGAG cally much more concentrated than an equivalent lysate
prepared from cultured mammalian cells. Lysate prepared from
3. The ribosome profiling protocol 50 to 100 ml of yeast liquid culture at an OD600 of 0.60.7 will
have 5 to 10-fold higher RNA concentration than lysate pre-
3.1. Preparation of a cell lysate pared from adherent cultured mammalian cells according in
Section 3.1.2.
3.1.1. Liquid cultures of the budding yeast Saccharomyces cerevisiae
3.1.1.1. Yeast cell harvesting. 3.1.2. Adherent cultured mammalian cells
1. For each culture to be harvested, prepare an open 50 ml conical This portion of the protocol was developed using HEK293 cells
tube full of liquid nitrogen in a rack held in a bath of liquid (ATCC, CRL-1573). Optionally, an experimenter may wish to treat
nitrogen. Punch several holes in the cap of the conical tube with cells with inhibitors of translation before harvesting. Several
a needle and set it aside. authors have observed that treatment of cells with chemicals that
2. To rapidly harvest, filter the yeast from the culture onto a inhibit initiation (harringtonine & lactimidomycin) results in the
0.45 lM Whatman membrane using a vacuum filter apparatus. accumulation of ribosome footprints around the start codon,
We use the Kimble Kontes ULTRA-WARE microfiltration assem- enabling the genome-wide identification of translation start sites
bly with fritted glass support (see Table 1). [5,13,17,18]. We have seen good results with a 2 min treatment
3. Immediately scrape the yeast from the filter with a straight- with 2 lg/ml harringtonine in DMSO at 37 C [5,13]. A different
edge metal spatula and plunge it into the conical tube of liquid approach is been taken by Qian and colleagues: either a 30 min
nitrogen. Use a second chilled metal spatula to dislodge the treatment with 50 lM lactimidomycin in DMSO at 37 C (termed
yeast from the straight-edge spatula. For ribosome profiling GTI-seq [18]); or cell lysis in the presence of 5 lM lactimidomycin,
we routinely use 150 ml cultures of haploid yeast at an OD600 followed by a puromycin treatment to remove non-initiating ribo-
of 0.60.7. This volume of culture produces an excess of cell somes (termed QTI-seq [17]).
lysate, but is used because smaller cultures form a very thin Routine pre-treatment of cells with the elongation inhibitor
layer on the filter, which is difficult to scrape off effectively. cycloheximide before harvest is now thought to excessively skew
4. Drip 2 ml of lysis buffer (see Section 2.1.2) into the liquid nitro- the natural distribution of elongating ribosomes (Weinberg et al.
gen. Adding the lysis buffer drip-wise is necessary so that fro- [19] provides a thorough discussion of this issue). However, inhibi-
zen droplets are small enough to fit inside the mixer mills tion of elongation prior to cells lysis may still be desirable for
grinding jars. The working volume for the 10 ml grinding jars specific experiments. We have found that a 1 min treatment with
that we typically use for yeast lysis is 24 ml, but the grinding 100 lg/ml cycloheximide in DMSO at 37 C is effective.
jars are available in different volumes.
5. Cap the tube and place it upright in a 80 C freezer until the 1. Aspirate media from one 10 cm dish of adherent cells. Place the
liquid nitrogen evaporates (typically 30 min to an hour). Frozen dish on ice, gently wash with 5 ml ice-cold PBS, and aspirate the
yeast can be stored indefinitely at 80 C. Be sure to always use PBS thoroughly.
a pierced cap to close liquid nitrogen filled tubes! 2. Perform in-dish lysis: drip 400 ll ice-cold lysis buffer (see Sec-
tion 2.1.2) onto the cells, taking care to cover the entire surface
of the dish.
3.1.1.2. Yeast cell lysis. 3. Tip the dish and scrape cells down the slope into the lysis buffer
1. For each culture, open a mixer mill grinding jar and chill all its pooled in the lower corner. Pipette lysis buffer from this pool
components, including the grinding ball, in liquid nitrogen. back towards the top of the dish and scrape again down the
2. Add frozen yeast to the grinding jar, insert the grinding ball, and slope of the dish.
seal it tightly. Chill the assembled chamber in liquid nitrogen. 4. Pipette the cells in lysis buffer and withdraw the entire contents
3. Loosen the grinding jar about one quarter turn, place it in the of the dish to a microfuge tube on ice. Pipette several times to
mixer mill, and grind yeast for 3 min at 15 Hz. Tighten the disperse cell clumps and incubate for 10 min on ice.
grinding jar and return it to liquid nitrogen. Repeat the grinding 5. Triturate cells ten times through a 26 gauge needle.
for 5 more cycles of 3 min each. 6. Clarify the lysate by centrifugation for 10 min at 20,000g, 4 C
4. Chill the grinding jar, along with two rounded metal spatulas, in and recover the supernatant. If you are not proceeding directly
liquid nitrogen. to nuclease footprinting, flash freeze the lysate by immersion in
5. Partly fill an open conical tube with liquid nitrogen and place it liquid nitrogen, and store indefinitely at 80 C.
upright in the liquid nitrogen bath. Open the grinding chamber
and recover the powder into the conical tube of liquid nitrogen 3.2. Nuclease footprinting and ribosome recovery
using the chilled metal spatulas, re-chilling them as needed.
6. If you are using a given cup to grind more than one sample, be Nuclease digestion conditions have a large impact on the suc-
sure to wash the cup thoroughly between samples to avoid cess of a ribosome profiling experiment. Experimenters should
cross-contamination. proceed with caution: the two commercial suppliers of E. coli
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8 N.J. McGlincy, N.T. Ingolia / Methods xxx (2017) xxxxxx
RNase I use different unit definitions to measure RNase I activity, place it in a sideways microfuge tube rack, and allow it to air-
and substantial changes in the RNase activity during nuclease dry for 10 min. Resuspend the RNA in 5 ll 10 mM Tris pH 8.
footprinting could compromise the experiment. Moreover, unpub- RNA may be stored overnight at 20 C or for months at 80 C.
lished reports have suggested that optimizing digestion conditions
(particularly, decreasing the amount of RNase I) on a case-by-case
basis can often be advantageous. Indeed, empirically we have 3.3. Footprint fragment purification
found that matching the RNA-to-RNase ratio controls better for
footprint size than matching RNase concentration alone. Critically, the electrophoresis apparatus used for this and
Before proceeding with nuclease footprinting a portion of the subsequent preparative RNA gels must be maintained free of RNase
undigested cell lysate is reserved for the preparation of an RNA- contamination. Decontaminate the tank and electrodes if the
seq library. We typically use Illuminas Tru-seq protocol according equipment has been used for other purposes. Molecular biology
to the manufacturers instructions, and employ an rRNA depletion grade water obtained directly from the purifier can be tested for
step. This RNA-seq library provides estimates of mRNA abundance nuclease contamination and used to prepare running buffer due
that are used for the calculation of translational efficiency (see to the large volume of nuclease-free water needed for this purpose.
Section 3.11.1). While ribosome footprints were initially characterized to be
28 nt fragments, and this remains the modal footprint size, a
1. Determine the RNA concentration of the cell lysate. We class of so-called small footprints of 20 nt were discovered by
typically use the Quant-iT RiboGreen assay according to the Lareau et al. in 2014 [20]. The current protocol has been modified
manufacturers instructions, measuring blue fluorescence with to capture both small and large footprints, hence the recom-
a GloMax-Multi Jr Single Tube Multimode Reader and mended size range of 1734 nt for excision from the footprint frag-
accompanying Fluorescence Optical KitBLUE (see Table 1). ment purification gel. If only large footprints are desired, oligos NI-
Cell lysates typically need to be diluted 1:1001:10000 in order 800 & NI-801 should be used as a guide to excise the region from
to be quantified using RNA standards in the range of 1 ng/ll 2634 nt from the gel.
20 pg/ll.
2. Take lysate containing 30 lg total RNA as determined in step 1. Pre-run a 15% polyacrylamide TBE-Urea gel for 15 min at
3.2.1 and dilute to 200 ll with polysome buffer (see Table 2). 200 V in 1X TBE.
Add 1.5 ll RNase I (10 U/ll by the Epicentre definition) and 2. Add 5 ll 2X denaturing sample loading buffer (see Table 3)
incubate for 45 min at room temperature with gentle agitation. to each RNA sample. Prepare a control oligo sample for
3. Add 10 ll SUPERase*In RNase inhibitor to stop nuclease diges- two lanes consisting of: 1 ll lower marker oligo NI-800
tion, transfer the digestion to ice. Our experience has indicated 10 lM, 1 ll upper marker oligo 10 lM, 8 ll 10 mM Tris pH
that if the digestion is promptly cooled and spun, SUPERase*In 8, and 1 ll 2X denaturing sample loading buffer. Prepare a
addition isnt necessary to stop the digestion. ladder sample with 0.5 ll 10 bp ladder 1 lg/ll, 4.5 ll
4. Transfer the digestion to a 13 mm 51 mm polycarbonate 10 mM Tris pH 8, and 5 ll 2X denaturing sample loading
ultracentrifuge tube and underlay 0.9 ml sucrose cushion by buffer.
carefully positioning a pipette tip (or a cannula or similar tool) 3. Denature samples for 90 s at 80 C, then load them onto the
at the very bottom of the tube and slowly dispensing the polyacrylamide gel. Load the control oligo sample on either
sucrose solution. The lysate should float on top of the sucrose, side of the RNA samples. Additionally, in order to facilitate
leaving a visible interface between the layers. the isolation of the small footprints described by Lareau
5. Pellet ribosomes by centrifugation in a TLA100.3 rotor at et al. [20], load 12 ll of NEB miRNA marker in similar
100,000 rpm, 4 C for 1 h or 70,000 rpm for 2 h. The TLA110 positions.
(8-tube) rotor can also be used. Previous versions of the proto- 4. Separate by electrophoresis for 65 min at 200 V.
col used longer centrifugation times, however the conditions 5. Stain the gel for 3 min with 1X SYBR Gold in 1X TBE running
presented here are more than sufficient to pellet 80S particles. buffer on a gentle shaker.
In performing sedimentation time calculations, scale by 4.3X 6. Visualize the gel; for each footprinting sample, excise the 17
for 34% sucrose and further 1.5X for 4 C versus 20 C. nt 34 nt region demarcated by the lower band of the
6. Mark the outside edge of the ultracentrifuge tube, where the miRNA marker and the upper size marker oligo (see Fig. 2).
ribosome pellet will be found, before removing the tube from This will result in the isolation of both large and small foot-
the rotor. Gently pipette the supernatant out of the tube. The prints. Place each excised gel slice in a clean non-stick
ribosomal pellet is glassy and translucent, and may not be vis- RNase-free microfuge tube.
ible until the supernatant is removed. Resuspend the ribosomal 7. Extract RNA from the polyacrylamide gel slices. Add 400 ll
pellet in 300 ll TRIzol reagent or equivalent. RNA gel extraction buffer (see Table 4) and freeze samples
7. Purify RNA from the resuspended ribosomal pellet using the for 30 min on dry ice.
Direct-zol kit according to the manufacturers instructions for 8. Leave samples to thaw overnight at room temperature with
purifying total RNA including small RNAs. Collect the eluate in gentle mixing on a nutator.
a non-stick RNase-free tube. Critically, from this point until 9. Briefly centrifuge gel extractions to collect the liquid at the
the end of the reverse transcription reaction, proper techniques bottom of the tube. Transfer the 400 ll eluate into a clean
must be employed to avoid RNase contamination. This includes non-stick RNase-free microfuge tube.
the rigorous use of gloves and RNase-free reagents and 10. Precipitate the extracted RNA by adding 1.5 ll GlycoBlue,
consumables. mixing, and then adding 500 ll isopropanol. Precipitations
8. Precipitate RNA from the eluate by adding 38.5 ll water, 1.5 ll may be left at 20 C overnight. Recover RNA as described
GlycoBlue, and 10 ll 3 M NaOAc pH 5.5, followed by 150 ll iso- in step 3.2.9.
propanol. Chill precipitation for 1 h on ice. Precipitations may 11. Resuspend the size-selected RNA in 4 ll 10 mM Tris pH 8
be left overnight at 20 C. and transfer to a clean non-stick RNase-free microfuge tube.
9. Pellet RNA by centrifugation for 30 min at 20,000g, 4 C in a RNA may be stored overnight at 20 C or indefinitely at
tabletop microfuge. Carefully pipette all liquid from the tube, 80 C.
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Table 10
Composition of the T4 PNK end-healing reaction.
Table 11
Composition of the linker ligation reaction.
we estimate that >80% of RNA fragments become ligated to a deadenylase. L 10 bp ladder. The 100 nt band and selected other bands of the
linker. 10 bp ladder is indicated on the right-hand side of the gel. Ligated product is
0
indicated. The effect of Yeast 5 deadenylase and RecJ treatment on the pre-
3. To specifically deplete unligated linker from the ligation
adenylated linker band is indicated by the box.
reaction before reverse transcription, add 0.5 ll Yeast
5 -deadenylase 10 U/ll and 0.5 ll RecJ exonuclease 10 U/ll
0
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10 N.J. McGlincy, N.T. Ingolia / Methods xxx (2017) xxxxxx
reduction in the pre-adenylated linker band, but the intensity of of Gerashchenko & Gladyshev [23], but systematic studies of these
the ligation product band is not affected. parameters are lacking. We have heard discussion of authors plan-
ning hybrid approaches: first employing commercial rRNA deple-
3.4.1. Pooling and purification of ligations tion reagents, and then using subtractive hybridization to target
Purify ligations on an Oligo Clean & Concentrator column, particular problem fragments. Indeed, this could be achieved using
which is necessary to recover sequences of this length. Before the biotinylated rRNA depletion oligos described in Ingolia et al.
purification, one may pool ligations with different sample barcodes [13]. An interesting alternative approach is suggested by Faridani
in order to multiplex those samples for sequencing. et al., who employed a masking oligo to prevent linker ligation to
the abundant 5.8S rRNA [24].
1. Each sample to be pooled will contribute 11 ll volume. Calcu-
late the total volume of the pool as 11 ll x the number of 3.6. Reverse transcription
samples.
2. Take Oligo Binding Buffer equal to twice the total sample vol- 1. In order to assess the performance of your reverse transcription
ume and add the samples to be pooled, along with any supple- reaction, prepare three control samples: 1. a positive control
mentary water, into the binding buffer. For example, with six containing 10 ll of ligation control oligo (NI-805) at 100 nM,
samples (66 ll) use 132 ll Oligo Binding Buffer. Mixing samples diluted from a 1 lM stock in 10 mM Tris pH 8; 2. a no-insert
in the denaturing oligo binding buffer likely guards against any control containing 10 ll of a linker at 100 nM; 3. a no-
cross-sample ligation by undigested linkers and residual ligase template control containing 10 ll 10 mM Tris pH 8.
activity. 2. Add 2 ll reverse transcription primer (NI-802) at 1.25 lM to all
3. Add ethanol equal to 8 times the total sample volume. For samples and controls. Denature for 5 min at 65 C in a thermal
example, with six samples (66 ll total sample volume) use cycler and then place on ice. Cool the thermal cycler to 50 C.
528 ll ethanol. 3. Set up the reverse transcription reaction tabulated in Table 12
4. Load samples onto the Zymo Spin-Column nested in a collection and incubate for 30 min at 50 C in the thermal cycler.
tube. Load no more than 800 ll (corresponding to 8 samples) at We have observed that the yield of full-length RT product
once repeat loading and spinning if the total volume exceeds appears constant between 47 C and 54 C in this reaction, but
800 ll at this point. the activity of untemplated nucleotide addition disappears at
5. Centrifuge the column for 30 s at 12,000g and discard the flow- 50 C (see Fig. 4). However, when using SuperScript III, untem-
through. plated nucleotide addition persists through 52 C but is lost at
6. Add 750 ll DNA Wash Buffer, centrifuge the column for 30 s at 55 C; and reverse transcription yield did not decline at 57 C,
12,000g, and discard the flow-through. the highest temperature tested. We recommend performing
7. Centrifuge again (with no wash) for 1 min at maximum speed to reverse transcription at 55 C when using SuperScript III.
remove any residual wash buffer. 4. Hydrolyze the RNA template by adding 2.2 ll 1 M NaOH to each
8. Transfer the Zymo-Spin Column into a new microcentrifuge reaction and incubating at 70 C for 20 min.
tube and add 26 ll RNase-free water. Centrifuge for 30 s at 5. Add 28 ll water to the reverse transcription reaction, bringing
12,000g and recover RNA in the eluted liquid. N.B. If no rRNA the volume to 50 ll, and purify the sample using an Oligo Clean
depletion will be performed, elute directly in 10 ll RNase-free & Concentrator kit according to the manufacturers instructions,
water and proceed to reverse transcription (Section 3.6). Other- except performing the final elution in 6 ll nuclease-free water.
wise, RNA may be stored overnight at 20 C, or indefinitely at 6. Separate the reverse transcription products from the unex-
80 C. tended primer and no-insert background products by polyacry-
lamide gel electrophoresis as described in Section 3.3, using 6 ll
3.5. Ribosomal RNA depletion 2X denaturing sample loading buffer with each 6 ll reverse
transcription product. Omit the preparation of marker oligo
Perform Ribo-Zero Gold depletion on the purified linker-ligated samples and instead prepare one sample containing 2 ll reverse
footprint fragments from step 3.4.1.8 according to the manufac- transcription primer at 1.25 lM, 3 ll 10 mM Tris pH 8, and 5 ll
turers protocol, using the larger volume of rRNA Removal Solution 2X denaturing sample loading buffer.
(suitable for up to 5 lg of total RNA). Omit the 50 C incubation 7. Excise the reverse transcription product bands from the gel and
immediately prior to bead removal: advice from Illumina is that place each in a clean non-stick RNase-free microfuge tube. A
the rRNA fragments present after nuclease footprinting are quite typical gel is shown in Fig. 5. The reverse transcription primer
short, and the 50 C incubation is too stringent for efficient deple- is 57 nt long (evident from the no-template control, or the
tion. Recover RNA using an Oligo Clean & Concentrator column reverse transcription primer only sample, see Fig. 5, lanes 1 &
according to the manufacturers instructions, eluting the purified 2), the no-insert reverse transcription product is 76 nt long (evi-
RNA in 10 ll RNase-free water at the final step. The ligated foot- dent from the no-insert control comprising only the linker, see
print fragments are quite short and thus the depleted RNA must Fig. 5, lane 3), and the full-length reverse transcription product
be recovered by an approach that captures small RNAs, such as is ~105 nt long (the centre of mass of the sample reverse tran-
the Oligo Clean & Concentrator column. RNA may be stored at scription product should migrate at the same size at the ligation
20 C overnight or indefinitely at 80 C. control, NI-805, compare Fig. 5, lanes 5 & 6). The DNA linker is
We are aware that many authors find a significant proportion of
their ribosome profiling library to consist of rRNA derived Table 12
sequences, even when some form of rRNA reduction is employed. Composition of the reverse transcription reaction.
Anecdotally, we see greater persistence of rRNA sequences in situ- Component Volume (ll) Final
ations where rRNA depletion reagents are older, where there is RNA sample & primer 12 NA
more starting material, and where RNase I digestion might have 5X Protoscript II buffer 4 1X
been more severe. This might suggest some combination of greater dNTPs 10 mM ea. 1
damage to the ribosome producing more smaller rRNA fragments 0.1 M DTT 1 5 mM
SUPERase*In (20 U/ll) 1 20 U
that either overwhelm the rRNA depletion reagents, or escape
Protoscript II (200 U/ll) 1 20 U
them all together. This conjecture is consistent with the results
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N.J. McGlincy, N.T. Ingolia / Methods xxx (2017) xxxxxx 11
Fig. 4. Hotter reverse transcription reduces untemplated extension without reducing yield. Reverse transcription of ligated footprints (left), linker only (middle), or RT primer
alone (right) with Protoscript II at temperatures ranging from 46 C to 57 C. Reaction were resolved by polyacrylamide gel electrophoresis as described in Section 3.3. An
example of non-templated addition to the reverse transcription (RT) primer is indicated. L 10 bp ladder, the 100 nt band and selected other bands are indicated on the left-
hand side of the gel.
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12 N.J. McGlincy, N.T. Ingolia / Methods xxx (2017) xxxxxx
3.8. qPCR quantitation of circularized cDNA For example, if the input circles are present at 700 pM, then
2.3 ll circles in a 50 ll PCR will give 32 pM of template for a
qPCR quantitation of circularization reactions is used to deter- 12-cycle amplification. Ensure that the template comprises no
mine the optimal conditions (amount of circularized template more than 10% of the final PCR reaction, ideally no more than
and number of cycles) for a single library construction PCR. 5%. This strategy ensures that the primer concentration remains
greater than 10 times the concentration of extended PCR pro-
1. Prepare a dilution series of the NI-803 positive control. Prepare duct, and thus that in later cycles primer annealing and exten-
a 10 nM working solution by diluting 2 ll of a 1 lM stock solu- sion predominates over re-annealing of the two template
tion in 198 ll 10 mM Tris, pH 8. Dilute 10.2 ll of the 10 nM strands. The cycle numbers given in Table 15 derive from
working solution with 89.8 ll 10 mM Tris, pH 8 to construct a empirical calibration of qPCR-quantified input template with
1.02 nM stock solution. Serially dilute 2 ll of this stock into the appearance of the unwanted, re-annealed duplexes in the
6 ll of 10 mM Tris, pH 8 to prepare a 1:4 dilution (256 pM), a final gel.
1:16 dilution (64 pM), a 1:64 dilution (16 pM), and a 1:256 dilu-
tion (4 pM). 3.9. Library construction PCR
2. Determine the number of qPCR reactions. This should include
one for each point on the standard curve, as well as two blanks 1. For each sample pool, prepare a 50 ll PCR according to Table 16.
(one using nuclease-free water and one using 10 mM Tris, pH Use a different reverse indexing primer for each sample pool
8), two NI-804 negative controls (with 1 nM template), and that will be combined into a single sequencing lane. Prepare a
two replicates of each circularization reaction (diluted up to positive control reaction using synthetic control oligo NI-803,
10-fold for samples expected to be high-concentration). One diluted from the 10 nM stock to achieve the same template con-
can also include replicates of circularization reactions per- centration as the actual circularization reactions. The forward
formed on NI-804, in order to assess the efficiency of circular- primer is NI-798. Reverse primers are NI-799, 822826 (see
ization. Multiply the number of qPCR reactions by 20 ll, and Section 2.2.5).
then add an extra 10%. Prepare the common qPCR mixture 2. Perform PCR amplification with an initial denaturation for 30 s
according to the Table 14. at 98 C followed by 716 cycles (see Section 3.8) of 10 s denat-
3. For each reaction, combine 19 ll of qPCR mix with 1 ll uration at 98 C, 10 s annealing at 65 C, and 5 s extension at
template. 72 C. Finish with a 5 min extension at 72 C.
4. Perform qPCR amplification using the following cycling condi- 3. Purify PCR products using a DNA clean & concentrator column
tions: 15 min at 95 C, followed by 40 cycles of 10 s at 94 C, according to the manufacturers instructions. Use a 5: 1 ratio
20 s at 54 C, 30 s at 72 C, and plate reading. Follow the PCR of DNA binding buffer to sample, that is, 250 ll of DNA binding
amplification with a melt curve analysis. buffer for a 50 ll PCR. Elute into 25 ll 10 mM Tris, pH 8.
5. Fit a standard curve to the Cq values for the NI-803 dilution 4. Add 5 ll 6X non-denaturing sample loading buffer to each reac-
series. The 1.02 nM sample should have a Cq of roughly 8. Verify tion (see Table 6). Prepare a ladder sample with 1 ll 10 bp lad-
that the NI-804 negative control and the blank reactions have der, 11.5 ll 10 mM Tris pH 8, and 2.5 ll 6X non-denaturing
Cq values much higher than the standard curve or library circu- sample loading buffer.
larization samples. The NI-804 negative control will likely show 5. Set up a pre-cast 8% polyacrylamide non-denaturing gel. Load
a Cq value corresponding to 1 pM, with a broad melt curve. two adjacent wells with 15 ll each of the purified PCR samples,
6. Determine the template concentration in the circularization as well as loading one well with 15 ll 10 bp ladder.
reactions using the standard curve from the NI-803 dilution ser- 6. Separate PCR reactions by electrophoresis in 1X TBE running
ies. Select the amount of template and the number of cycles for buffer for 40 min at 180 V. Stain the gel for 3 min in 1X SYBR
the library construction PCR. The template concentrations in Gold in 1X TBE gel running buffer.
Table 15 give the final concentration of circles in a 50 ll PCR. 7. Visualize the gel and excise the amplified PCR product; a typical
gel is show in Fig. 6. Full-length libraries are 160 bp long,
whereas no-insert products are 136 bp long and unextended
Table 14 RT primer products are 117 bp long. Avoid the lower part of
Composition of the qPCR reaction. the product band, which is derived from unextended reverse
Component Volume per 100 ll (ll) Final (after template) transcription primer. Place excised gel slices in clean, non-
2X qPCR Master Mix 50.0 1X stick microfuge tubes.
100 lM NI-827 0.5 500 nM 8. Recover DNA from the gel slices as described in Section 3.3. It is
100 lM NI-828 0.5 500 nM particularly important that the gel extractions remain at 25 C
Nuclease free water 44.0 NA or below to avoid the formation of re-annealed partial duplexes.
Such duplexes will complicate the quantitation of the library.
Precipitations may be stored indefinitely at 20 C.
Table 15
PCR cycles required to amplify given concentrations of circular library cDNA.
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N.J. McGlincy, N.T. Ingolia / Methods xxx (2017) xxxxxx 13
Fig. 7. Library length distribution analysis using the Agilent 2200 TapeStation. An electropherogram image of the length distribution of the ribosome profiling library
generated by the TapeStation analysis software. Note the single major peak at 168 bp, within the range of what we typically expect for a ribosome profiling library. The small
proportion of larger sequences, and the absence of a discernable no-insert product peak are also fairly typical. Insert: The false gel image generated from the same sample.
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14 N.J. McGlincy, N.T. Ingolia / Methods xxx (2017) xxxxxx
Table 17 Table 19
Initial read processing fates for a MiSeq sequenced ribosome profiling library. Footprint fragment count by alignment fate.
linker (see Section 2.2 and Fig. 1). In analysing the composition of Our procedure now becomes increasingly specific to ribosome
the UMI, we have found that there is often a preference for A & G in profiling. A desired length interval (typically 1636 bp for libraries
0 0
the 5 N of NI-802, the nucleotide that becomes ligated to the 3 end that include small footprints) of the filtered alignments are inter-
of the cDNA footprint during circularization. The absence of a dis- sected with BED file based annotation for protein-coding tran-
cernable correlation between the identity of this nucleotide and scripts from the organism of interest. We can then examine the
that of the footprint nucleotide it becomes ligated to suggests that frequency distribution of alignment lengths, and meta-gene pro-
0
changing the NN at the 5 end of NI-802 to RN may make circular- files of the areas around start and stop codons. Such analyses
ization more efficient. Lastly, sequences are assigned to new FASTQ determine whether the aligned fragments meet our expectations
files based on the sample barcode in their linker, and discarded if for ribosome footprints. An example of such an analysis for the
this barcode is unspecified, or cannot be determined. Typical sample barcoded ATCGT is shown in Fig. 8. This sample was pre-
results of such splitting for a library comprising five samples is illu- pared from wild-type S. cerevisiae BY4742 grown to OD600 0.6 in
strated in Table 18. YEPD. Despite the relatively low number of fragments considered,
Each sample specific FASTQ file is then aligned against a variety the frequency distribution of fragment lengths shows the expected
of ncRNA sequences (e.g. rRNAs, snoRNAs, snRNAs etc.) using Bow- major peak at 28 nt, representing large footprints, and a smaller
tie2 [25]; sequences that align are removed. The majority of these peak at 20 nt, representing small footprints (Fig. 8A). Examining
0
ncRNA-aligning sequences align to rRNAs. Indeed, in the absence of the distribution of fragment 5 ends around start codons (Fig. 8B),
rRNA depletion, most of the sequences in the library will align to one can appreciate the enrichment of fragments at the start codon
rRNA. When rRNA depletion is effective, we have found that the by the large peak 12 nt upstream. We can also observe the
proportion of sequences that align to rRNA depends on the biolog- expected enrichment of fragments within the ORF compared to
0
ical source the cell lysate used for footprinting. In our hands, the 5 UTR, and the three nucleotide periodicity in frame prefer-
libraries constructed from cultured mammalian cells typically con- ence. The heatmap in Fig. 8C breaks down the picture from
tain 30% rRNA-aligning sequences after effective rRNA reduction, Fig. 8B by fragment length, illustrating the clear dependence of
while libraries constructed from cultures of S. cerevisiae typically periodicity on fragment length. The same picture for the region
contain as much as 70% rRNA-aligning sequences. However, these around the stop codon is shown in Fig. 8D. Both Figs. 8C & D illus-
proportions can vary widely (see Section 3.5). trate interesting enrichments in certain fragment lengths over start
The remaining sequences, that do not align to ncRNAs, are then and stop codons.
aligned against the transcriptome of interest using Tophat2 [26]. To measure the degree to which a transcript is translated we
From these alignments, those that are of the desired quality are count the number of footprints aligning to the central portion of
extracted for further analysis. At this point we utilize the extended the ORF, by convention 15 codons from the start codon until 5
UMI to remove sequences that are likely to represent duplicates codons before the stop codon. We focus on this area of the ORF
generated by the library construction PCR when multiple because the ribosomes there are the most likely to be undergoing
sequences with the same alignment share the same extended elongation, and therefore their frequency should reflect the trans-
UMI, one sequence is selected arbitrarily, and the others discarded. lational capacity of the transcript rather than the kinetics of initia-
The number of fragments with each of these alignment fates is out- tion and termination. In addition, we impose two further
lined in Table 19. A number of important points are evident: first conditions to increase the likelihood we are counting genuine foot-
that rRNA reduction worked poorly in this particular experiment, prints: first, we only count fragments of lengths that represent >5%
but enough useful alignments remain to draw useful conclusions of total fragments. Secondly, in order to determine whether frag-
about the library. Secondly, that the rate of sequence duplication ments spanning the boundary between the central ORF and the
by the library construction PCR is low. After this filtering the areas around the start & stop codons should be counted, we deter-
sequence alignments for useful fragments are stored in indexed, mine for each fragment the likely location of the ribosomal A-site
sorted BAM files, which can be browsed manually by the Integrative the site of aminoacyl-tRNA decoding. If the fragments inferred A-
Genomics Viewer (IGV, [27,28]), if desired. site is within the central portion of the ORF, the fragment is judged
to be the footprint of a ribosome undergoing elongation, and thus
0
counted. The offset between the 5 end of the fragment and the
ribosomal A-site is inferred for each fragment length representing
Table 18 >5% of total fragments by examining the frame bias of fragments of
Read fates after splitting on sample barcode. that length at the start codon. Presuming variance in the digestion
0
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N.J. McGlincy, N.T. Ingolia / Methods xxx (2017) xxxxxx 15
Fig. 8. Characteristics of ribosome footprints. All panels are based on the analysis of a single sample from the described library (barcode ATCGT), prepared from wild-type S.
cerevisiae BY4742 grown to OD600 0.6 in YEPD. A The frequency distribution of fragment lengths between 1636 nucleotides (nt). B A meta-gene plot of fragments of all
0 0
lengths whose 5 end aligns within 100 nt of the start codon. The height of each bar represents the number of fragment 5 ends aligning to a given nucleotide position. The
color of the bar represents the frame of that nucleotide relative to the A in the AUG start codon. The lower panel illustrates the portion 5585 nt after the start codon with
0
greater resolution. C & D are heatmaps illustrating the log10 of the number of fragment 5 ends (after the addition of a pseudo count of 1) of each fragment length at each
nucleotide around the start codon (C) and stop codon (D).
of ribosome profiling specific software tools have emerged. Many In attempting to determine which transcripts are being differ-
researchers are interested in being able to determine which tran- entially translated between two or more conditions, ribosome pro-
scripts are differentially translated between two or more condi- filing suffers from the same analytical problems that afflict all
tions. This requires that we also take into account any changes in genome-wide measurements. Of particular note are the assump-
the mRNA level (typically measured by RNA-seq from the same tions that underlie between-sample normalization of sequencing
sample), which will also effect the number of ribosome footprints depth: that the per-cell amount of material isolated from each
obtained from a transcript. While we routinely utilise the R library sample is approximately the same, that the majority of genes do
DESeq2 [29] for this task, three groups have developed specific sta- not change in expression, and that the proportion of genes that
tistical models to call differential translation taking into account are up-regulated and the proportion that are down-regulated are
data from both ribosome profiling and RNA-seq measurements: approximately equal [3335]. In situations where we observe glo-
anota [30], RiboDiff [31] & Xtail [32]. bal unidirectional changes in expression, such as treatments that
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16 N.J. McGlincy, N.T. Ingolia / Methods xxx (2017) xxxxxx
cause a global decrease in translation, these assumptions lead to As we have alluded to, many other authors have adopted the
inaccurate quantification [3335]. This problem can be remedied ribosome profiling procedure and developed it for their own pur-
by the use of a spike-in control, a strategy that has received consid- poses. These developments range broadly from changes necessary
erable attention in the development of RNA-seq [36]. A known to apply the method to other organisms, to the enzymology of
amount of a synthetic RNA is included early in library preparation; library preparation, to fundamental changes in the method of ribo-
the signal from this synthetic RNA is then used to normalize the some isolation. In subsequent paragraphs, we aim to summarize
signal between samples. To date, this approach is evident in only these innovations, presenting the choices and challenges they offer
a limited number of ribosome profiling studies, each of which to a newcomer to the procedure.
use a different spike-in control: Han et al. [37] used a single syn- Depending on the experiment in question, choice of nuclease
thetic 28 nt RNA sequence [37], while Popa et al. fragmented a may be a pertinent issue. The protocol outlined in this article
longer synthetic RNA to an average length of 30 nt [38]. An alter- focuses on the digestion of lysates made from S. cerevisiae cultures
native approach was taken by Iwasaki et al. [10], who normalized and adherent mammalian cell-lines with RNase I. RNase I was ini-
expression using the sum of the fragments aligning to mitochon- tially chosen because of its robust activity in S. cerevisiae cell
drial transcripts. Mitochondrial transcripts are translated by a dis- lysates, and its lack of a discernible nucleotide preference for diges-
tinct mitoribosome, which is also captured by the ribosome tion, but other nucleases can also be used. In particular, due to the
profiling protocol [39]. Translation by the mitoribosome is not inactivity of RNase I in E. coli lysates, micrococcal nuclease is com-
effected by the translation inhibitor rocaglamide A, which enabled monly used in E. coli ribosome profiling experiments [4951].
Iwaskaki et al. to employ this normalization strategy to examine Micrococcal nuclease has also be utilized in the ribosome profiling
the determinants of the global decrease in translation caused by of Drosophila melanogaster derived samples. While Drosophila pro-
rocaglamide A [10]. In contrast to RNA-seq, we are not aware of filing libraries have been made using RNase I [52], Drosophila ribo-
any systematic examination of the relative merits of different somes are highly sensitive to damage by RNase I, and micrococcal
spike-in controls for ribosome profiling. nuclease has worked better for some authors [53]. Of particular
Besides those aimed at differential expression analyses, a interest in this regard is a recent study by Gerashchenko & Glady-
diverse set of other software tools exist for ribosome profiling. shev [23], who examine the use of several different nucleases to
They each provide a different series of methods for dealing with produce ribosome profiling libraries from several different species.
ribosome profiling data more generally including tasks such as Gerashchenko & Gladyshev observe that while the number of foot-
the alignment & counting footprints, and the derivation of sum- prints aligning to a given gene is correlates well between libraries
mary statistics such as the frequency distribution of footprint constructed using different nucleases, the footprint distributions
lengths and genome-wide codon occupancy. Spread across com- along a given mRNA are quite different. They suggest that both
puting platforms, they include two R packages (riboSeqR [40] & the identity and amount of nuclease should be optimized based
RiboProfiling [41]), a Galaxy based webserver (RiboGalaxy [42]), on the species under examination. However, some of the nucleases
a Python library (Plastid [43]), and stand-alone software (Ribomap they examine have pronounced sequence specificities for cleavage
[44]). Lastly, two software tools seek rigorous methods to identify (e.g RNase A & RNase T1), which could make framing information
translated RNA sequences using ribosome profiling data: RiboTa- more difficult to deduce from such libraries.
per employs a spectral analysis of ribosome profiling data to pro- There are now four methods by which digested monosomes are
vide identification of translated ORFs based on the three- isolated: first, ultracentrifugation through a sucrose cushion as
nucleotide periodicity observed in ribosome footprints [45,46], described in Section 3.2; secondly, ultracentrifugation through a
whereas Rfoot rather does the opposite: using the absence of sucrose gradient followed by the specific isolation of monosomes
three-nucleotide periodicity to identify footprints generated from using a gradient station; thirdly, through size-exclusion chro-
the protection afforded by non-ribosomal RNPs to ncRNAs matography (SEC) spin columns; and fourthly, by pull-down of a
(sequences which are typically discarded in the analysis of ribo- molecular handle associated with the ribosome. Methods that
0
some profiling libraries), and mRNA 3 UTRs [47]. employ pull-down can be set apart from the others they have
typically been designed to isolate and profile ribosomes from
specific cell-types [54,55], and specific locations within the cell
4. Conclusions [56,57]. While they are the basis of the TruSeq Ribo Profile kit from
Illumina, we are aware of only a limited number of studies that
In conclusion, ribosome profiling gives us unprecedented power have employed SEC spins columns to isolate digested monosomes
to explore not only the mechanisms of translation, but also its reg- [39,5861]. The majority of ribosome profiling studies employ one
ulation in vivo. As one might expect from something of such inter- of the two ultracentrifugation based methods. In comparing the
est and complexity, the ribosome profiling protocol is under two methods, ultracentrifugation through a sucrose gradient fol-
constant development, both in our laboratory and others. Here lowed by the specific isolation of monosomes using a gradient sta-
we have outlined the current state of our ribosome profiling proto- tion is the only way to ensure that nuclease digestion results in
col, noting improvements we have made since the publication of complete conversion of polysomes to undamaged monosomes.
Ingolia et al. [13], and illustrating its ability to produce a library Moreover, this procedure is also likely to result a more stringent
that shows all the characteristics of ribosome footprints. purification of monosomes away from the diverse set of mRNPs
Ribosome profiling is not without its limitations. Perhaps its which likely pellets with monosomes upon ultracentrifugation
most fundamental of which is that the snap-shot of through a sucrose cushion. This being said, the sucrose cushion
transcriptome-wide ribosome location it provides is by definition procedure was developed to overcome specific issues with the
static. As a result we cannot differentiate a translating ribosome sucrose gradient procedure, mainly that it is slow and technically
from a static ribosome in a single sample. To some extent, this lim- challenging. This makes it more difficult to scale, and increases
itation can be circumvented by multi-sample experiments: for the likelihood that the distribution of ribosomes will change from
example, the progressive depletion of footprints from ORFs, and that present at the moment the sample was harvested. We are also
their concurrent accumulation around start codons, observed dur- aware of at least one study that has dispensed with ribosome iso-
ing a timecourse of harringtonine treatment in mammalian cells lation altogether, extracting RNA from digested lysate and pro-
[5,48]. ceeding directly to an RNA fragment size-selection gel [62].
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N.J. McGlincy, N.T. Ingolia / Methods xxx (2017) xxxxxx 17
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