Yarden Katz
RIBO-SEQ CHECKLIST
Starting from FOOTPRINT RNA after ribosome pellleting and RNA extraction.
(A) Dephosphorylation:
1. ___ Take 9.5 ul of RNA to PCR tube.
2. Make sample mix for five samples:
___ 2.5 ul RNAseIn
___ 6.25 ul 10x PNK buf
___ 6.25 ul PNK enzyme
3. ___ Add 3 ul of sample mix per sample
4. ___ Incubate 1 hour at 37 C.
(B) 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
(C) Subtractive hybridization (BEFORE polyA-tailing)
1. ___ Transfer 10 ul of size-selected footprint RNA to PCR tubes
2. ___ Make RiboSub Mix (assume 6 samples):
___ 120 ul RiboSub Primer Mix (10pM)
___ 30 ul RNase H buffer
___ 60 ul water
3. ___ Add 35 ul RiboSub Mix per sample
4. ___ Denature RNA/oligo mix at 72 C for 2 mins
5. ___ Add 1 ul RNAse-inhib. per sample
6. ___ Incubate at 37 C for 30 mins
7. ___ Add 2 ul RNase H per sample
8. ___ Incubate at 37 C for 15 mins
9. ___ Add 2 ul TuboDNAse per sample
10. ___ Incubate at 37 C for 15 mins
11. ___ Transfer samples to Eppys
12. ___ Add 50 ul water per sample (brings vol up to one hundred)
13. Phenol extract each sample:
___ 100 ul ACID phenol
___ Spin 30 mins at 4 C
___ Take supernatant to new tubes
14. Ethanol precipitate each sample:
___ Add 10 ul NaOAc
___ Add 250 ul 100% EtOH
___ Add 2 ul Glycogen
15. ___ Let sit on dry ice for ~20 mins
16. ___ Spin 30 mins at 4 C
17. ___ Wash with 500 ul 70% EtOH
18. ___ Spin 20 mins at 4 C
19. ___ Let pellet airdry
20. ___ Resuspend in 15 ul 10mM Tris pH 7.0 (or 30 ul, take 15 for backup),
prepare for polyA step
(D) 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)
- ___ Add 7.5 ul water per sample (brings volume up to twenty two point
five ul
- ___ Transfer to PCR tubes
- Prepare 2X tailing mix (assuming 8 samples):
___ 40 ul PAP buffer
___ 40 ul ATP
___ 120 ul H2O
- Prepare enzyme mix (assuming 8 samples):
___ 20 ul 2x tailing mix
___ 16 ul H2O
___ 4 ul PAP enzyme (polymerase)
- ___ Denature RNA samples 2 min at 80C in thermocylcer, then place on ice.
- ___ Add 22.5 ul 2X tailing mix
- ___ Add 5 ul enzyme mix, see below, on ice (final volume 50 ul)
2. ___ Incubate 10 min at 37 C. Transfer samples to Eppys.
3. ___ Add 200 ul 5 mM EDTA to quench the reaction.
4. ___ Add:
___ 2 ul glycoblue
___ 28 ul 3 M NaOAc
___ 300 ul isopropanol. S
5. ___ Set at -20 C to chill (20 mins or more)
6. ___ Spin 30 min at max speed, 4 C.
7. ___ Wash with 0.5 ml 70% EtOH at -20 C.
8. ___ Spin for 30 min at 4 C, max speed.
9. ___ Resuspend total RNA sample in 12 ul 10 mM Tris 8.0 (prepare for RT)
(E) Reverse transcription
1. ___ Get 25 uM RT primer (10 ul 50uM primer in 10ul H20, or 5 uL 100uM
primer in 15 uL H20)
2. ___ Thaw DTT / FS reagents
3. ___ Transfer 11.5 ul each sample to PCR tubes
4. Set up the sample reaction mix (assuming 6 samples)
___ 6 ul dNTPs (10mM)
___ 6 of RiboRT primer (20 uM)
5. ___ Add 2 ul sample reaction mix per sample
6. ___ Denature at 65 C for 5 min. Place on ice.
7. ___ Prepare 1st strand mix:
___24 ul FS Buffer
___ 3 ul RNAseInhib.
___ 6 ul 0.1M DTT
___ 6 ul SuperScript III enzyme
8. ___ Add 6.5 ul 1st strand mix per sample
9. ___ Incubate 30 min at 48 C.
10. ___ Add 2.1 ul 1 M NaOH to each RT reaction.
11. ___ Incubate for 15 min at 98 C.
12. ___ Neutralize with 2.1 ul 1 M HCl.
13. 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.
14. ___ Pre-run a 10% TBE urea gel and pre-run at 200V.
15. ___ Denature samples 1 min at 95 C and load. You will need to load 2
lanes/sample.
16. ___ Run gel 65 min at 200 V.
17. ___ Stain 5 min in SYBR Gold 1:10,1000 in TBE.
18. ___ Excise extended RT product band (should be ~30 bp longer than RT
primer band).
19. ___ Recover nucleic acids as described in SI. Nucleic acids recovery, but can
incubate at room temperature for cDNA extraction.
20. ___ Resuspend pellets following RT product gel extraction in
15 ul 10 mM Tris pH 8.0
(F) Circularization of ssDNA
1. ___ Take 15 ul of samples to PCR tubes
2. Make circularization mix (6 samples):
___ 12 ul CircLigase buf
___ 6 ul ATP
___ 6 ul MnCl2
3.
4.
5.
6.
7.
___ Add 4 ul circularization mix to each sample and mix well.
___ Add 1 ul circ Ligase enzyme 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.
(G) PCR Amplification for sequencing
1. ___ Prepare 5 uM stocks of RP / RPI primers (make FWD + RPIN mixes).
The FWD primer is from Yevgenia (YLK003)
2. ___ Perform PCR for 6, 8, 10 cycles (assume ~4 cycles)
3. ___ Prepare the sample PCR mixes. Prepare a master mix for each sample
assuming 4 cycles. Example:
___ 13.36 ul 5X HF Buf
___ 1.34 ul 10mM DNTPs
___ 6.72 ul RPI N mix (use the appropriate primer per sample!)
___ 46.8 ul H2O
4.
5.
6.
7.
___ 0.67 ul Phusion Polymerase enzyme
___ Add 4 ul of circularized DNA sample to the appropriate sample’s mix (mix
well!)
___ Add 16.7 ul of the PCR mixed with the sample’s DNA to PCR tubes
___ Perform PCR
___ Run RiboSeq PCR (take out samples after 6, 8, 10 cycles):
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)
8. ___ Pull out a tube for the library after cycle 6, 8, 10, and 12. Place tubes on
ice.
9. ___ 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)
10. ___ Run on 8% TBE gel for 40 minutes at 200 V. (NOT urea)
11. ___ 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.
12. ___ Extract DNA from the gel slices.
13. ___ Resuspend pellet in 10 ul 10 mM Tris 8.
14. ___ Give it to the BMC for Solexa sequencing.
15. ___ Get a beer/skim latte.
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 SpinX 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 70 % EtOH at -20 C.
NOTE: Combine pellets if you have split tubes from gel extraction!
10. ___ Air dry and resuspend in the specified buffer.