Yarden Katz
Adapted from Dan Treacy, Eric Wang (Burge Lab @ MIT) and Mary-Kay
Thompson (Gilbert Lab @ MIT)
Protocol from Musashi-genes.org
Isolate ribosome footprints from cells and tissues
This procedure involves isolating ribosome footprints by pelleting of ribosomes.
Lyse cells and treat with RNase
1. Treat trypsinized cells in a conical tube with 100 ug/mL cycloheximide for 5
minutes @ 37 deg
2. Spin down cells, wash in PBS with 100 ug/mL cycloheximide, spin again, and
then lyse in lysis buffer with cycloheximide (~2 mL per 10 cm plate).
o Lysis buffer:
o 20 mM HEPES pH 7
o 100 mM KCl
o
o
o
o
o
3.
4.
5.
6.
7.
8.
5 mM MgCl2
0.5% Na Deoxycholate (Add H2O before this!)
0.5% NP-40 (Glycogen)
1 mM DTT
protease inhibitors (1 tablet / 10 ml)
Lyse on ice for ~10 minutes.
Treat with 10 uL Turbo DNase and 2 uL NEB RNase I (all amounts per 2 mL), 5
minutes at 25 deg with shaking on rotator
Spin for 10 minutes @ 17K (or max speed) @ 4 deg. Transfer to green 2mL epp
tubes.
Save supernatant and discard pellet
Add more RNase I and treat for 55-75 minutes at 25 deg (amount should be
scaled to OD260 of supernatant, approx. 8 uL for 2 mL of lysate where OD260 on
nanodrop is ~10). Add more, e.g. 5 uL for 10 mL. Blank with Lysis Buffer.
Freeze supernatant at -80 deg, or proceed with ribosome pelleting
Pellet ribosomes and isolate RNA
1. Rinse ultracentrifuge tubes in handsoap/water. Turn on ultracentrifuge. Cool rotor
in cold room prior to spin. Pour 10 mL lysis buffer into centrifuge tube.
2. Use a 10 mL pipette to layer sucrose cushion below lysis buffer layer. Add 12.5
mL sucrose cushion.
o Sucrose cushion:
o 20 mM HEPES pH 7
o 100 mM KCl
o
o
5 mM MgCl2
0.5 M Sucrose
3. Pipette lysate on top. If needed, add more lysis buffer so that meniscus is above
where tube narrows.
4. Make sure tubes are balanced: weigh them and add Lysis buffer if needed.
5. Spin at 60K for 1 hour 50 minutes in 70Ti.
6. Pour off supernatant. Look for translucent pellet at bottom of tube.
7. Add 500 uL of 8M Guanidium HCl and vortex the pellet off the tube. Transfer the
pellet and broken up particles to a 1.5 mL eppendorf.
8. Vortex on Noah’s shaker at 4 degrees until pellet is dissolved (~20 minutes).
9. Add 700 uL 25:24:1 [Phenol Chloroform]. Vortex, let phases separate, and spin at
14K at 4 deg for 10 minutes.
10. Split tubes in 2 tubes per sample.
11. Transfer aqueous phase to new tube, add 3M Na Acetate, linear
acrylamide/glycogen, and 100% EtOH for standard precipitation. Example for
350 uL:
o 1/10x of Na Acetate: 35 uL
o Acrylamide: 2 uL
o 2.5x of 100% EtOH: 875 uL
12. Freeze at -80 or -20 overnight. Spin at 17K for 25 minutes at 4 degrees to pellet
RNA.
13. Wash pellet with 70% EtOH, spin again (15 mins/17k rpm), dry pellet, and
resuspend in 20 uL Tris 10 mM pH 7.0.
Before dephos step, split each sample in 2 tubes.
Gilbert lab ribosome profiling (modified from Ingolia et. al, 2009)
Last mod. 1/13/10, MKT
I.
Growing the cells:
1. Inoculate 6 ml YPAD culture and grow overnight (> 16 hrs)
2. Dilute to an OD600 of 0.001 in 750 ml pre-warmed YPAD and grow overnight to
an OD between 0.6-0.7 (takes ~18 hrs)
II.
Extract preparation:
1. Prepare aliquot of polysome lysis buffer to use the day of with:
0.1 mg/ml CHX___
NO DTT ___
NO RNaseIN__
(DTT and RNaseIN inhibit RNaseI activity for footprinting!)
2. Grow 750 ml yeast culture to OD600 = 0.6-0.7
3. Add cycloheximide to final concentration of 0.1 mg/ml. Put cultures back on the
shaker for an additional 2 minutes
4. Harvest cycloheximide-treated cells: spin 7000 rpm in JLA8.100 rotor at 4 C for 5
min in pre-chilled large centrifuge bottles.
5. Resuspend pellet in 40 ml cold lysis buffer and transfer to 50 ml falcon tube,
keeping everything on ice. Pellet cells for 5 min at 3500 rpm in table-top
centrifuge. Wash cell pellet with 30 ml cold lysis buffer and resuspend by
vortexing. Pellet cells for 5 min at 3500 rpm in table-top centrifuge. Remove all
supernatant and weigh cell pellets.
6. For each gm of pellet weight, resuspend cell pellets in 1.5 ml of 1ysis buffer and 5
gm of glass beads.
7. Lyse cells by vortexing 6 x 20 seconds, incubate on ice for at least 30 seconds
between cycles.
8. Spin crude extract at 3500 rpm for 5 min in table top. Take off supernatant to
Oakridge tubes, and spin in SS-34 (Sorvall) at 4oC for 20 min at 11.500 rpm.
9. Remove extract to new tube, avoiding pellet and top lipid layer. Determine A260
of extract with nanodrop.
10. Make aliquots of 55 OD.
11. Flash freeze aliquots of extract in liquid nitrogen and store at –80oC.
III.
Footprinting and monosome isolation
1. Pre-chill sw41 rotor and buckets at 4 C.
2. prepare aliquot of 10% and 50% polysome gradient buffer to use the day of with:
0.1 mg/ml CHX___
3 mM DTT ___
NO RNaseIN__
3. Thaw extract aliquots on ice and setup RNaseI digestion with 50 OD units of
extract plus RNase I (15U/A260 unit). (i.e. 7.5 ul from a 100 U/ul stock)
4. Incubate reactions in Thermomixer at 25 C, 400 rpm for 1 hour and keep on ice
after finishing until ready to load gradients.
5. Setup gradients by filling tubes with 10% sucrose polysome gradient buffer and
uderlaying 50% sucrose polysome gradient buffer.
6. Cap the gradients and use the Gradient Master to form the sucrose gradients by
tilted tube rotation. (Program= 10-50% long) long= cap size. Chill at least 20
minutes before loading samples.
7. Load the samples of cell extract on top of gradients. Spin gradients 3 hours at
35000 rpm, 4 C in ultracentrifuge.
8. Return buckets to 4 C for storage during fractionation.
9. Set UV monitor to max range (AUFS= 2). Set dist= 98, Num= 1, Speed= 0.2
(mm/sec). Dataq program: file-> record. Autozero on rinse before fractionation.
10. Fractionate the digested sample to capture the prominent monosome peak, which
should occupy a volume of a bit over 2 mls, collect in Oakridge tubes. I just
collect 70 second fractions after the slope change from 60S to 80S.
11. Proceed immediately to RNA extraction from collected fractions.
IV.
RNA extraction:
i) RNA extraction for total RNA library:
1. To 600 ul of extract: add 40 ul 20% SDS and 650 ul acid phenol. Vortex.
2. Incubate 10 min at 65 C in thermomixer at max speed (1400 rpm).
3. Rest 5 min on ice.
4. Add 650 ul chloroform to pre-spun 1st phase-lock (2 ml size). Add the sample to
the tube and invert to mix.
5. Spin at top speed (13.2k g) for 5 min at RT.
6. Add 650 ul phenol:chloroform:iaa to prespun 2nd phase-lock
7. Transfer supernatant from 1st phase-lock to 2nd phase-lock.
8. Mix and spin 5 min at max speed.
9. Add 650 ul chloroform to same phase-lock. Mix by inverting.
10. Spin at top speed for 5 min.
11. Remove aqueous supernatant and add 1/9 volume 3M NaOAc, pH 5.5.
12. Add 1 volume isopropanol. Mix and chill at least 30 min at -20 C.
13. Spin at max speed, 4 C, 30 minutes, to pellet RNA.
14. Wash pellet in 0.75 ml 80% EtOH at -20 C.
15. Air dry and resuspend in 100 ul 10 mM Tris 7.5 (to prepare for polyA selection).
ii) RNA extraction for footprinting library
1. Split collected fraction into aliquots of 0.6 ml and put in 2 ml tubes.
2. Add 40 ul 20% SDS and 650 ul acid phenol. Vortex.
3. Incubate 10 min in 65 C water bath, vortex every minute.
4. Rest 5 min on ice.
5. Add 650 ul chloroform and vortex. Spin at top speed (13.2g) for 5 min.
6. Remove aqueous supernatant to a new tube and add 650 ul phenol:chloroform:iaa.
Vortex.
7. Spin at top speed for 5 min.
8. Remove supernatant and add 650 ul chloroform. Vortex and spin at top speed for
5 min.
9. Remove aqueous supernatant and add 1/9 volume 3M NaOAc, pH 5.5.
10. Add 1 volume isopropanol. Mix and chill at least 30 min at -20 C.
11. Spin at max speed, 4 C, 30 minutes, to pellet RNA.
12. Wash pellet in 0.75 ml 80% EtOH at -20 C.
13. Dry and resuspend in 0.5 ml 10 mM Tris 8 (combine the 3 tubes/monosome
fraction here). Add 1.25 ul RNAseIn.
14. Load onto YM-100 microconcentrator and spin 55 min at 500xg, 4C.
15. Recover the flow-through (~ 400 ul) and add 1/9 volume 3M NaOAc, 2 ul
glycoblue and 1 volume isopropanol. Mix and chill at least 30 min at -20 C.
16. Spin 30 min at top speed, 4 C to pellet RNA.
17. Wash pellet in 0.75 ml 80% EtOH at -20 C and airdry.
18. Resuspend in 10 ul 10 mM Tris 7 (to prepare for dephosphorylation).
V. Preparing total RNA fragments for library construction:
i) Poly-A selection from total RNA:
- Prepare sample:
1. 200 ug of sample= ___ ul
2. Bring up to 100 ul with 10 mM Tris 7.5.
3. Add 100 ul binding buffer.
4. Heat 2 minutes at 80 C at 400 rpm in the thermomixer.
5. Place on ice and add 0.5 ul RNasin Plus.
- Prepare beads:
1. Resuspend oligodT beads by vortexing.
2. Take 200 ul resuspended beads to tube on magnet. Remove storage buffer.
3. Wash 1X with 100 ul binding buffer.
4. Add 100 ul binding buffer to resuspend beads.
-Perform purification:
5. Add denatured RNA to tube with beads and binding buffer.
6. Incubate 5 min at RT with rotation.
7. Put beads on magnet and remove supernatant.
8. Wash 2x with 200 ul wash buffer B.
9. Add 16 ul of 10 mM Tris 7.5 and heat at 80 C, 400 rpm in thermomixer, for 2 min to
elute.
10. Remove elute from beads to new tube on ice.
ii) Total RNA fragmentation:
1. Setup fragmentation reaction in PCR tube on ice:
16 ul purified RNA (ideally your whole output from the poly-A selection)
4 ul 5X RNA fragmentation buffer (Ambion)
2. Incubate reactions in PCR machine 5 min at 94 C.
3. Immediately place on ice, add 2 ul 10X stop solution and mix well. Keep on ice.
4. Add 80 ul H20, 11 ul 3M NaOAc, 2 ul glycoblue and 100 ul isopropanol.
5. Chill at least half an hour at -20 C.
6. Spin 30 min at 4 C at max speed.
7. Wash with 0.75 ml 80% EtOH at -20 C.
8. Dry and resuspend in 10 ul 10 mM Tris 7.0
*From this point on, total RNA and footprint samples processed in parallel (except for
subtractive hybridization protocol, which is only performed on the footprint samples)
Yarden Katz
Modified November 12, 2011
START HERE AFTER RIBOSOME PELLETING
V. Dephosphorylation:
1. Take 9.5 ul of RNA to PCR tube.
2. Add: 0.5 ul RNaseIn (SUPERase In), 1.25 ul 10X PNK buffer, 1.25 PNK. (Final vol:
12.5 ul)
3. Incubate 1 hour at 37 C.
VI. Size selection (Use 2 gels for four samples, separate lanes out far!)
1. Set up 15% TBE/Urea/polyacrylamide gel (BioRad, precast), pre-run 20 min at
200V.
2. Add 12.5 ul 2X denaturing loading dye to each dephosphorylation.
3. Prepare 10 bp ladder, marked 10/60 oligo standard [single-stranded] (and 100 bp if
you want): 1 ul ladder, 9 ul water, 10 ul 2X loading dye.
4. Prepare control 28mer to mark desired size: 1ul 50 uM 28mer, 9ul water, 10 ul 2X
denaturing loading dye.
5. Denature samples 2 min at 75 C, then keep on ice until loading.
6. Load gels and run 65 min at 200V (Bromophenol blue will be at bottom).
7. Stain gels 5 min in SYBR Gold (1:10,000 in 0.5X TBE or RNase free water).
8. Photograph gel, before cut.
9. Excise region in the sample lane corresponding to the size of the control 28mer. (See
gel pictures for example)
10. Photograph gel again after cut.
11. Excise the 28mer band to use as a control in downstream reactions.
12. Extract RNA as described in S1.
13. Resuspend in 20 ul 10 mM Tris pH 7.0. Use 10 ul for subtractive hyb.
14. Make a dilution to submit to the biomicrocenter for quantification on the small RNA
chip before proceeding to library prep (a 1:50 dilution of your sample will likely be
sufficient; 0.5 ul RNA, 9.5 ul RNAse-free water).
15. PERFORM SUBTRACTIVE HYBRIDIZATION, BEFORE POLYA-TAILING
VIII. Subtractive hybridization
1. Perform subtractive hybridization according to Gloria Brar’s protocol.
2. Resuspend in 12 l 10mM Tris pH 7.0 to prepare for polyA tailing / RT step.
VII. Poly-A tailing:
1. Sample preparation:
-10 pmol total mRNA = ____ ul
-10 pmol fp RNA = _15_ ul (use all of sample from subtractive hyb which was
resus in 15 ul)
- bring to 22.5 ul with water and put in PCR tubes
2. Denature RNA samples 2 min at 80C in thermocylcer, then place on ice. Add
22.5 ul 2X tailing mix and 5 ul Enzyme mix, see below, on ice (final volume 50 ul)
2X tailing mix: (total 25 ul/sample, but only add 22.5 ul to each tube):
5 ul 10X PAP buffer
5 ul 10 mM ATP (1 mM ATP final conc.)
0.75 ul RNAse In (30 Units)
14.25 ul H20
Enzyme mix: (total 5 ul/sample):
2.5 ul 2X tailing mix (from above)
2 ul H20
0.5 ul PAP enzyme (5 U/ul)
3.
4.
5.
6.
7.
8.
9.
Incubate 10 min at 37 C. Transfer samples to Eppys.
Add 200 ul 5 mM EDTA to quench the reaction.
Add 2 ul glycoblue, 28 ul 3 M NaOAc, and 300 ul isopropanol. Set at -20 C to chill.
Spin 30 min at max speed, 4 C.
Wash with 0.5 ml 80% EtOH at -20 C.
Spin for 15 min at 4 C, max speed.
Resuspend total RNA sample in 12 ul 10 mM Tris 8 (prepare for RT)
IX. Reverse transcription
1. Set up the sample reaction:
11.5 ul resuspended template
1 ul dNTPs (10 mM)
1 ul oNTI225 (50 uM)
2. Denature at 65 C for 5 min. Place on ice and add:
4 ul First-strand buffer (5X)
0.5 ul RNAseIn Plus
1 ul DTT (0.1 M)
1 ul SuperScriptIII
3. Incubate 30 min at 48 C.
4. Add 2.1 ul 1 M NaOH to each RT reaction.
5. Incubate for 15 min at 98 C.
6. Neutralize with 2.1 ul 1 M HCl.
7. Prepare to load on gel by adding 22.2 ul 2X denaturing loading buffer to sample.
Prepare ladders as before. Also prepare the RT primer to run on the gel: 0.5 ul RT
primer at 50 uM, 9.5 ul H2O, and 10 ul denaturing loading dye.
8. Pre-run a 10% TBE urea gel and pre-run at 200V.
9. Denature samples 1 min at 95 C and load. You will need to load 2 lanes/sample.
10. Run gel 65 min at 200 V.
11. Stain 5 min in SYBR Gold 1:10,1000 in TBE.
12. Excise extended RT product band (should be ~30 bp longer than RT primer band).
13. Recover nucleic acids as described in SI. Nucleic acids recovery, but can incubate at
room temperature for cDNA extraction.
X. Circularization of ssDNA
1. Resuspend pellets following RT product gel extraction in 15 l 10 mM Tris pH 8.
2. Make circularization mix:
2 ul 10X circLigase buffer
1 ul 1 mM ATP
1 ul 50 mM MnCl2.
3.
4.
5.
6.
7.
Add 4 ul circularization mix to each sample and mix well.
Add 1 ul circ ligase to each sample and mix well.
Incubate at 60 C for 60 minutes.
Incubate at 80 C for 10 minutes to heat inactivate.
Store reaction at -20 C or proceed directly to amplification.
X1. PCR Amplification for sequencing
1. Prepare the PCR mastermix (1X shown below. You will do 4 rxns/library):
3.34 ul 5X HF buffer
0.334 ul 10 mM dNTPs
0.84 ul 100 uM oNTI230
0.84 ul 100 uM oNTI230
11.7 ul water
0.167 ul Phusion (2U/ul)
2. Take 4.5 ul of circularized sample to tube and add mastermix for 4.5 reactions. Mix
well.
3. Prepare 4 PCR tubes (separate) and pipette 16.7 ul of the reaction into each tube.
4. Run RiboSeq PCR:
1) 98 C, 30 sec
2) 98 C, 10 sec
3) 60 C, 10 sec
4) 72 C, 5 sec
5) Repeat 2-4 11X (i.e. 12 cycles total)
5. Pull out a tube for the library after cycle 6, 8, 10, and 12. Place tubes on ice.
6. Prepare add 3.4 ul 6X DNA loading dye to reactions. Load 1 ul 10 bp ladder (1ul
ladder, 15.7 ul water, 3.3 ul 6X loadying dye)
7. Run on 8% TBE gel for 40 minutes at 200 V.
8. Cut out the bands at 120 bp. Do not cut from saturated lanes or ones with high
molecular weight products. Make sure that you get good separation from the 90 bp
primer band and be sure not to cut any of this out.
9. Extract DNA from the gel slices.
10. Resuspend pellet in 10 ul 10 mM Tris 8.
11. Give it to the BMC for Solexa sequencing.
12. Get a beer.
S1. Nucleic acid extraction
1. Prepare an extraction tube by piercing a 0.5 ml tube with an 18.5 gauge needle and
place inside a 1.7 ml tube.
2. Spin the nested tubes 3 min at 20,000 xg to force the gel slice through the needle hole
into the larger tube.
3. Freeze gel pieces in -80 to help break up matrix (~30 min)
4. Soak gel in 0.4 ml of relevant elution buffer overnight with rotation. (For RNA, elute
in the cold room. For DNA, elute at RT)
5. Cut off end of p1000 pipette tip and use it to transfer gel to Corning Spin-X column
(transfer the gel/elution mixture to the filter of column)
6. Spin 3 min at 20,000 xg to recover the elution free of the gel debris.
7. Add 2.0 ul Glycoblue (15 mg/ml) and 0.44 ml isopropanol. Mix and precipitate at
least 30 min at -20 C.
8. Spin 30 min at 20,000 xg to pellet nucleic acids.
9. Remove supernatant and wash in 0.50 ml 80 % EtOH at -20 C.
10. Air dry and resuspend in the specified buffer.
Buffers
Polysome isolation buffers:
1X Polysome Lysis Buffer
20 mM Hepes-KOH, pH 7.4
2 mM Mg acetate
100 mM K acetate
0.1mg/ml cycloheximide (make fresh!)
1% Triton-X 100
1X Polysome Gradient Buffer
20 mM Hepes-KOH, pH 7.4
2 mM Mg acetate
100 mM K acetate
0.1 mg/ml cycloheximide (make fresh!)
3 mM DTT
oligodT dynabeads buffers:
Binding buffer
20 mM Tris-HCl, pH 7.5
1.0 M LiCl
2 mM EDTA
Wash buffer B
10 mM Tris-HCl, pH 7.5
0.15 M LiCl
1 mM EDTA
-you will also need 10 mM Tris-HCl, pH 7.5
nucleic acid elution buffers:
RNA elution buffer
300 mM NaOAc pH 5.5
1 mM EDTA
100 U/ml RNAseIn (add to aliquot at each use)
DNA elution buffer
300 mM NaCl
10 mM Tris pH 8.0
0 mM EDTA
Example gels
Size selection gels:
Total RNA size selection
Footprint size selection
Reverse transcription gel:
Extended product (cut)
Amplification for sequencing gel:
Insert with adapters
(cut)
le: 6Cyc
6
8
10
12
- Lanes 6 or 8 would be good to cut from. Lanes 10 and 12 are
over-saturated.
Technical notes:
IV. RNA extraction
i)
RNA extraction for total library:
- without phaselock tubes, it will be difficult to remove the first aqueous layer
because of the huge amount of protein that comes out of solution.
ii)
RNA extraction for footprinting library:
- I do this protocol for each footprint equivalent- i.e. split each monosome fraction
to 3 tubes then recombine later after spinning down in isopropanol. Because
fractions are cleaner than extract, this can be done with or without phase-locks)
V. Preparing total RNA fragments for library construction:
Poly-A selection from total RNA:
- Be sure to allow adequate time for beads to come out of solution. 1-2 minutes is
recommended by Invitrogen. I usually shorten this, but make sure the solution is
clear.)
VI. Size selection:
- I run a separate gel for every library b/c of potential contamination issues.
- I use a new stock of TBE + SYBR Gold for each staining and don’t re-use it on
other libraries.
- I put the gel on Syran wrap on the transilluminator to keep it clean.
- Glycoblue absords quite strongly at 260 nm so you cannot accurately use the
nanodrop to check the concentration of your RNA after this
VII. Poly-A tailing:
- Pavan and I have been starting with 5 pmol for the total RNA library. This is
because we have not been getting enough yield to make a 10 pmol library
even when we start from 200 ug of RNA. Considering that this is already 20
times more RNA than the Burge lab, for example, starts with, we think it is
sufficient.
VIII. Subtractive hybridization:
- only for ribosome footprinting library!
- Must only be done AFTER poly-A tailing.
- For source, see ribosomal RNA subtraction protocol (from Gloria Brar, UCSF,
attached at end of document)
IX. Reverse transcription:
- be sure to include a control oligo RT reaction (or at least run an old one on
your gel) to avoid ‘cutting blind’ in case you do not have enough RT product
on the gel to see. You will probably still have enough to sequence. In my
experience, you will have enough cDNA to see easily starting from 10 pmol of
RNA, but better to be safe).
X. Circularization of ssDNA
-
We have reason to believe that this step may not be as robust as some of the
others. It will help to avoid exposing circligase to higher than final reaction
salt conditions, etc., so make sure that the reaction is well-mixed before
adding enzyme.
Protocol changes/history:
1/12/10 (MKT)
differences from Ingolia protocol:
- polysome lysis and gradient buffer composition
- isolation of total RNA from lysis extract rather than cells
- use of salt fragmentation instead of alkaline fragmentation (we use Ambion’s
RNA fragmentation reagents)
- Poly-A tailing done in 50 ul final volume instead of 20 ul to deal with salt
sensitivity of reaction (as recommended by Nick Ingolia)
- use of RNAseIn instead of SuperaseIn RNAse inhibitor (same number of units
used when possible, but where this results in an unpipettable amount added, the
number of inhibitor units might be changed (only really in dephosphorylation,
where we use 20 U instead of 10 U.
1/27/10 (MKT)
- resuspend total RNA extraction in 100 ul Tris 7.5 to make resuspension easier
- resuspend post polyAdenylation (for total library) and post-subtractive
hybridization (for footprint library) in 12 ul Tris 8 so that you don’t need to add
any water to the RT cocktail
- changed RT incubation time to 30 minutes as originally recommended by Nick
- Changed sequencing PCR recommendation to 4.5X mix to save more of the
circularized ssDNA in case the PCR needs re-doing.
- Changed sequencing PCR gel to 200V and 3.3 ul 6X loading buffer to add.