Brandon:LabNotes/Project1/2013-2-21
IVT round 5, frozen versus live cells[edit]
- IVT round 4 was able to show results are reproducible in terms of overlap with UCSC genome browser, and were almost better in this case with 80% overlap with RNase III generated data.
- For DNase I accessibility assays fresh cells are always used. This is how it has been done for DNase I assays, which are mainly used for DNase accessibility. According to this article, Rapid genome-scale mapping of chromatin accessibility in tissue several reasons why you need fresh cells and some drawbacks of DNase I accessibility assays.
- DNase I has narrow effective concentration window (thus need titrations etc)
- inhibited by high levels of actin, so each sample must be individually titrated.
- usually nuclei are isolated prior to DNase I digestion, since it reduced actin concentration.
- since nuclei need to be obtained fresh cells have to be used.
- Thus if the assay could be used on frozen cells, that would increase its ability to be used in a wider variety of contexts.
- In IVT R4 results Rnase III fragmentation seems better than Mg++ fragmentation. could try Mg++ fragmentation again in this round.
- testing on 1000 cell samples since that gives the best data
Why using RNAase III for fragmentation
- Rnase III creates 5'-PO4 and 3'-OH termini on the fragments
- however, Cuts ONLY dsRNA (used DNA blocking primer to block that)
- Preferentially cuts from 5’ and 3’ ends (used blocking primer)
- Rnase III results in 2 base 3’ overhangs
- Used in the generation of siRNAs for knockdown
- Mg++ fragmentation creates 5' OH and 3' PO4 termini, thus have to do end repair.
- End Repair with Shrink Akaline phosphatase, PNK, Antarctic phosphatase
- random nonamer is less selective and can prime off of more sequences
buffers compositions[edit]
1X T7 buffer: 400 mM Tris Hcl 8 mM MgCl2 2 mM spermidine-Hcl 25 mM NaCl PH 7.9 1X NEBNext RNase III Reaction Buffer: 10 mM Tris-HCl 10 mM Mg(Cl)2 1 mM DTT 60 mM NaCl pH 8.3 @ 25°C 10X Poly(A) Polymerase buffer: 500 mM Tris-HCl 2.5 M NaCl 100 mM MgCl2 pH 7.9 @ 25°C MMLV Invitrogen (though using clontech) 5X First-Strand Buffer 250 mM Tris-HCl (pH 8.3 at room temperature 375 mM KCl 15 mM MgCl2 0.1 M DTT
Before starting protocols[edit]
1. Check if have enough reagents etc for the protocol
- lysis buffer
- nextera transposomes
- transposase/transposome
- IVT reaction mixture
- cells etc
- blocking primer, RNase III, PolyA Polymerase, ATP, taq polymerase, other primers
2. Purify GM12878 DNA from GM12878 cells with DNeasy blood and tissue kit for cells (USE PROTEINASE K (invitrogen) AND protease (qiagen) AND RNAase A). quanititate DNA with nanodrop.
GM12878 DNA purification method used (followed DNeasy kit protocol for washes etc): a. pellet cells b. resuspend in: 1. 100 ul PBS 2. 10 uL proteinase K 3. 3 uL 5 AU Qiagen protease 4. 4 uL A797A promega RNase solution (RNase A) c. add 200 uL buffer AL, incuabate at 56C for 10 minutes d. step 3 in DNeasy kit protocol until finish
3. samples this time:
Samples: Index 49, N2 adaptor 1. Mg++, 1000 cells Index 50, N2 adaptor 2. Mg++, 1000 cells Index 51, N2 adaptor 3. Mg++, 1000 cells 1 hour freeze Index 52, N2 adaptor 4. Mg++, 1000 cells 1 hour freeze Index 53, N2 adaptor 5. Mg++, 5000 cells 1 hour freeze Index 54, N2 adaptor 6. Mg++, 1000 cells lysed without transposome complex IVT Index 55, N2 adaptor 7. Mg++, Nuclease free H20 only IVT Index 56, N2 adaptor 8. RNase III, 1000 cells Index 57, N2 adaptor 9. RNase III, 1000 cells Index 58, N2 adaptor 10. RNase III, 1000 cells 1 hour freeze Index 59, N2 adaptor 11. RNase III, 1000 cells 1 hour freeze Index 60, N2 adaptor 12. RNase III, 5000 cells 1 hour freeze Index 61, N2 adaptor 13. RNase III, 1000 cells lysed without transposome complex IVT Index 62, N2 adaptor 14. RNase III, Nuclease free H20 only IVT
IVT Protocol[edit]
- If need to make more transposome, do first 2 steps.
1. annealing of ME sequence to T7 transposon sequence
- a. Make 100 uM stock solution of T7tspn-top2 and T7tspn-bot.
- b. Incubate 5 uL of each oligo (100uM) with 40 uL EB buffer at 95C for 2 minutes. Oligo's now at 10 uM in 50 uL.
- c. cool to RT at 0.1 C/s
2. transposome complex generation, run controls!!!
- add the below components into one tube and incubate for 20 minutes at RT
1.25 uL of annealed transposon 1.25 uL of 100% sterile glycerol 2.50 uL of Ez-TN5 transposase
- store at -20, is good for a year
2.5 freeze cells
- aliquot cells into specific concentrations of 1000 cells/uL and 5000 cells /uL.
- 1 hour freeze samples put into -80 for 1 hour. Take out after 1 hour and let thaw to room temperature. Use cells in downstream procedure.
3. Prepare samples, lyse cells with lysis buffer
Samples: 1. 1000 cells 2. 1000 cells 3. 1000 cells 1 hour freeze -80 4. 1000 cells 1 hour freeze -80 5. 5000 cells 1 hour freeze -80 6. 1000 cells lysed without transposome complex IVT 7. Nuclease free H20 only IVT
- Make 10ml 10X lysis buffer (LB, 100mM Tris.Hcl pH 7.5, 100mM NaCl, 30mM MgCl2, 1% NP40, Crawford et al. PNAS 2003) in nuclease free H2O.
- Prepare aliquots of lymphocytes GM12878 (have GM20431, GM12878, MEFs) that contain 1000, 500, 100 cells.
- spin down cells to concentrate them as necessary.
- Prepare 2X LB from 10X buffer. mineral oil optional.
- if needed make 4 or 6 uL solutions. keep 1:1 ratio of cells:lysis buffer
- make each cellular concentration in triplicate for AluI amplification with PCR for cell number quantification! store samples and perform quantification later. Use pure DNA nanodropped controls!!
AluI PCR quant 5 uL AluI 245-263 primer 5 uL AluI 21-40 primer X sample X water _________ 25 uL
- incubate below mixtures at 37C for 30 mins.
' | 1. 1K IVT | 2. 1K IVT | 3. 1K IVT frze | 4. 1K IVT frze | 5. 5K IVT frze | 5. 1K lysed | 6. N-H20/PBS con |
cells | 1 ul | 1 ul | 1 ul | 1 ul | 1 ul | 1 ul | 1 PBS |
2X LB | 1 ul | 1 ul | 1 ul | 1 ul | 1 ul | 1 ul | 1 ul |
4. transposition reaction, using (T7tspn-top2)
- samples 6-7 get H2O instead of transposome
- add the below into one tube and incubate for 10 minutes at 55C.
1 uL nextera LMW buffer 2 uL lysed/pure genomic DNA (X ng/pg DNA) 1.2 uL Nuclease free water .8 uL prepared T7 transposomes (MAKE SURE TO ADD LAST) (if was proportional to shendure would use .625 uL) ___________ 5 uL total solution
method used in shendure paper: 4 uL nextera HMW buffer X uL genomic DNA at prepared quantities X uL Nuclease free water ______ 17.5 uL total solution add 2.5 uL of prepared transposomes
5. Protease digestion of transposase, protease inactivation
To each tube, add: 1 uL Qiagen Protease, for 5 uL reaction 1 uL of .5 for .1 AU final. (stock is 5 AU and diluted 10X. want .5 AU/uL final []) Incubate: 50C 10 minutes, 70C 20 minutes
6. Fill in reaction
- Add 6 uL 2X taq polymerase, run at 72C for 3 minutes. (same as nextera)
7. Maxiscript (Ambion) T7 Protocol, IVT
- DNA from PCR can be used directly in the MAXIscript Kit without any pretreatment or purification.
a. Thaw 10X Transcription Buffer and ribonucleotide solutions. Store the ribonucleotides (A, C, G, U) on ice, but keep 10X transcription buffer at room temp b. Assemble reaction mixture at room temperature, ADD IN ORDER AND MIX THOROUGHLY!!!! bring to 20 uL with Nuclease free water X uL DNA template (list 1 ug) 2 uL 10X Transcription Buffer 1 uL 10 mM ATP 1 uL 10 mM CTP 1 uL 10 mM GTP 1 uL 10 mM UTP 2 uL T7 Enzyme Mix b. Incubate reactions at 37C overnight for ~16 hours. (>10 uM limiting nucleotide)
8. Clean RNA with with Zymo RNA Clean & Concentrator-5 Kit. elute in 10 uL or less.
- can quantitate with Qubit or on TBU gel.
9. RNase III fragmentation (NEB), Mg++ fragmentation:
Mg++ fragmentation:
a. RNA fragmentation with MgCl and end repair with T4PNK X uL RNA (100ng-200 ng) .5 uL 10x Fragmentation buffer (or dilute to 5X and use 1 uL) 3-X uL Nuclease-free H2O __________ 5 uL Total b. Incubate at 94C for 5 min, place the tube on ice. Then perform the end repair as following: 5 uL RNA (100 ng total) .8 uL 10x Polymerase buffer (PAP buffer) .8 uL 10mM ATP 1.4 uL T4PNK enzyme ____________ 8 uL Total 37 C for 30 mins. c. proceed to PAP addition when ready.
RNase III fragmentation:
use 100-200 ng of RNA Starting Material: Purified mRNA (50–250 nanograms) 1. Add 5' end blocking DNA primer and do annealing to form DNA-RNA hybrid. *a. add 2 uL of blocking oligo (10 uM) to aliquot RNA sample that will be used in step 2. use T7-frag-block-top2 for T7-top2. T7-frag-block for T7-top3 *b. Incubate at RT for 5 minutes (mix well) 2. Mix the following components in a sterile PCR tube: a. 5 uL Purified mRNA + blocking primer (50-250 nanograms) .5 uL RNase III (1 unit/μl) .8 uL RNase III Reaction Buffer (10X) X uL Nuclease-Free Water add in DNA primer to protect 5' end since don't want degradation?? ____________ 8.5 uL total volume b. Incubate in a preheated thermal cycler for 5 minutes at 37°C. c. Heat inactivate RNase III at 65C for 20 minutes. d. Immediately cool on ice and proceed to PAP addition.
10. Poly(A) Addition with polyA polymerase (Enzymatics)
- enzymatics PolyA polymerase.
ALL RNA SAMPLES
a. to each each add: 1.2 uL polyA enzyme .8 uL 10 mM ATP ________ 10.5 uL (should be) b. Incubate at 37C for 10 minutes c. proceed immediately to Zymo cleaning.
11. Zymo RNA clean and concentrator cleanup.
- Resuspend in appropriate volume in nuclease free H2O (10 uL last time)
12. single strand synthesis MMLV RT (Clontech)
- Followed protocol for SMART MMLV Reverse Transcriptase
20 uL reaction 1. Add 2.5 uL 20 uM primer stock to RNA sample. Bring to final volume of 12.5 uL with Nuclease free H2O (one from BENG160 class, T20VN_PE_R) 2. heat the mixture to 70C for 3 minutes. Immediately cool on ice. 3. Add the following to the reaction. 2 uL 5X first strand buffer 2 uL dNTP mix 2 uL 100 uM DTT 1 uL N-H20 .5 uL SMART MMLV RT and mix (ADD LAST!!!!!) ____ 20 uL total 4. Incuvate at 42C for 60 minutes 5. Terminate the reaction by heating at 70C for 10 minutes
13. second strand synthesis (qPCR) (KAPA), addition of barcodes
Samples: Index 49, N2 adaptor 1. Mg++, 1000 cells Index 50, N2 adaptor 2. Mg++, 1000 cells Index 51, N2 adaptor 3. Mg++, 1000 cells 1 hour freeze Index 52, N2 adaptor 4. Mg++, 1000 cells 1 hour freeze Index 53, N2 adaptor 5. Mg++, 5000 cells 1 hour freeze Index 54, N2 adaptor 6. Mg++, 1000 cells lysed without transposome complex IVT Index 55, N2 adaptor 7. Mg++, Nuclease free H20 only IVT Index 56, N2 adaptor 8. RNase III, 1000 cells Index 57, N2 adaptor 9. RNase III, 1000 cells Index 58, N2 adaptor 10. RNase III, 1000 cells 1 hour freeze Index 59, N2 adaptor 11. RNase III, 1000 cells 1 hour freeze Index 60, N2 adaptor 12. RNase III, 5000 cells 1 hour freeze Index 61, N2 adaptor 13. RNase III, 1000 cells lysed without transposome complex IVT Index 62, N2 adaptor 14. RNase III, Nuclease free H20 only IVT
KAPA SYBR FAST qPCR mix until saturation, X35 cycles 12.5 uL KAPA SYBR FAST qPCR mix 2 uL primers, 2 uL F, 2 uL R (T7-top2-PCR-iaf or T7-top3-PCR-iaf) and (PCR_R.N2Ind[XX]) .5 uL H2O 10 uL DNA template (use half RT reaction) _____________ 25 uL KAPA SYBR cycles: 98C 3min, (98C for 30s, 60C for 30s, 72C for 2 min) X35, 72C for 5 min, 4C forever
- terminate before curves saturate (usually cycle 6-7)
14.qiaquick cleanup
- can quanitate with nanodrop
15. Gel Size selection
- gel size select from 400-800 bp, follow gel size selection protocol
- do not need to include controls.
16. Cloning and Transformation, then genewiz sequencing for verification of inserts
17. Submit for sequencing if genewiz sequencing checks out.
results[edit]
- RNA quantification results
ng/ul | total ng in 10 uL | ' |
146.2942541 | 1462.942541 | Index 49, N2 adaptor 1. 1000 cells |
163.2803366 | 1632.803366 | Index 50, N2 adaptor 2. 1000 cells |
97.9122542 | 979.122542 | Index 51, N2 adaptor 3. 1000 cells 1 hour freeze |
164.1758917 | 1641.758917 | Index 52, N2 adaptor 4. 1000 cells 1 hour freeze |
212.7219414 | 2127.219414 | Index 53, N2 adaptor 5. 5000 cells 1 hour freeze |
- amounts used for RNA fragmentation for 200 ng total of RNA for each sample.
for 200 ng/sample | ' | ' |
1.367107691 | uL | Index 49, N2 adaptor 1. 1000 cells |
1.224887235 | uL | Index 50, N2 adaptor 2. 1000 cells |
2.04264524 | uL | Index 51, N2 adaptor 3. 1000 cells 1 hour freeze |
1.218205657 | uL | Index 52, N2 adaptor 4. 1000 cells 1 hour freeze |
0.940194503 | uL | Index 53, N2 adaptor 5. 5000 cells 1 hour freeze |
- RNA after ~16.5 hours IVT, run on TBU gel
File:ZhangLab 2 2013-02-22 13hr 53min-labeled.jpg
- barcode addition PCR curves. terminated at end of 5th cycle since curves that went up were saturated. all RNase III samples "worked" while Mg++ samples did not "work". not sure why
- Curves are in order of sample listings below.
- Mg++ treated samples:
Samples: Index 49, N2 adaptor 1. Mg++, 1000 cells Index 50, N2 adaptor 2. Mg++, 1000 cells Index 51, N2 adaptor 3. Mg++, 1000 cells 1 hour freeze Index 52, N2 adaptor 4. Mg++, 1000 cells 1 hour freeze Index 53, N2 adaptor 5. Mg++, 5000 cells 1 hour freeze Index 54, N2 adaptor 6. Mg++, 1000 cells lysed without transposome complex IVT Index 55, N2 adaptor 7. Mg++, Nuclease free H20 only IVT
File:2013-02-21 round 5 fresh versus frozen mg++.bmp
- RNase III treated samples:
Samples: Index 56, N2 adaptor 8. RNase III, 1000 cells Index 57, N2 adaptor 9. RNase III, 1000 cells Index 58, N2 adaptor 10. RNase III, 1000 cells 1 hour freeze Index 59, N2 adaptor 11. RNase III, 1000 cells 1 hour freeze Index 60, N2 adaptor 12. RNase III, 5000 cells 1 hour freeze Index 61, N2 adaptor 13. RNase III, 1000 cells lysed without transposome complex IVT Index 62, N2 adaptor 14. RNase III, Nuclease free H20 only IVT
- FORGOT TO ADD IN SAMPLE 7 ON TEMPLATE, THUS MISSING. IS NTC AND SAMPLE 6 IS NTC AND WORKED SO IS FINE.
File:2013-02-21 round 5 fresh versus frozen rnIII.bmp
- gel quanitation after adding barcodes:
ng/ul | sample |
17.1224229 | Index 49, N2 adaptor 1. Mg++, 1000 cells |
14.9420755 | Index 50, N2 adaptor 2. Mg++, 1000 cells |
15.98039617 | Index 51, N2 adaptor 3. Mg++, 1000 cells 1 hour freeze |
15.68673145 | Index 52, N2 adaptor 4. Mg++, 1000 cells 1 hour freeze |
19.51780039 | Index 53, N2 adaptor 5. Mg++, 5000 cells 1 hour freeze |
10.50404988 | Index 56, N2 adaptor 8. RNase III, 1000 cells |
4.225618165 | Index 57, N2 adaptor 9. RNase III, 1000 cells |
2.789804401 | Index 58, N2 adaptor 10. RNase III, 1000 cells 1 hour freeze |
3.121451094 | Index 59, N2 adaptor 11. RNase III, 1000 cells 1 hour freeze |
4.118527799 | Index 60, N2 adaptor 12. RNase III, 5000 cells 1 hour freeze |
- TBE gels after barcode addition
- Mg++ samples:
File:ZhangLab 2 2013-02-25 11hr 47min-labeled.jpg
- Rnase III samples
File:ZhangLab 2 2013-02-25 11hr 52min-labeled.jpg
- input amounts for gel size selection
uL for 67 ng a sample | ' | ' |
6.378492178 | uL | Index 56, N2 adaptor 8. RNase III, 1000 cells |
15.8556683 | uL | Index 57, N2 adaptor 9. RNase III, 1000 cells |
24.01602061 | uL | Index 58, N2 adaptor 10. RNase III, 1000 cells 1 hour freeze |
21.46437602 | uL | Index 59, N2 adaptor 11. RNase III, 1000 cells 1 hour freeze |
16.26794895 | uL | Index 60, N2 adaptor 12. RNase III, 5000 cells 1 hour freeze |
ng/smpl | total ng RNIII | |
66.95530563 | 334.7765282 | |
for 67 ng a sample | ||
3.912997618 | uL | Index 49, N2 adaptor 1. Mg++, 1000 cells |
4.483982163 | uL | Index 50, N2 adaptor 2. Mg++, 1000 cells |
4.192636984 | uL | Index 51, N2 adaptor 3. Mg++, 1000 cells 1 hour freeze |
4.271125582 | uL | Index 52, N2 adaptor 4. Mg++, 1000 cells 1 hour freeze |
3.43276387 | uL | Index 53, N2 adaptor 5. Mg++, 5000 cells 1 hour freeze |
ng/smpl | total ng Mg++ | |
67 | 335 | |
104.2760123 | uL total all | |
- gel size selection R5
- was denser than thought based on quanitations above.
File:ZhangLab 2 2013-02-25 15hr 42min-labeled.jpg
- validation of gel size selection:
File:ZhangLab 2 2013-02-25 19hr 22min-labeled.jpg
UPDATE 3/22/2013
- ran on one lane of hi-seq at BIOGEM.
- gave different results for base composition and quality scores than usual libraries. For base composition, there are many more A's per cycle then other nucleotides. This is assumed to be because of the polyA issue where the read1 sequence is polyA'ed. Read qualitites drop off after 20 BP, but they are still phred scores above 30 which is high. maybe because of PCR
- base composition
- read qualities
future and conclusions[edit]
- blah balh
Blah