Brandon:LabNotes/Project1/2013-11-18
IVT Round 13, Replicate of GM12878 and 500 cells (brand new t7-top2 transposon), and tranposome dilution testing[edit]
- repeating 500 cell experiment so have replicates to do analysis on. can do correlations, and show that the same type of results are obtained.
- Doing transposome dilution testing, to see if less transposome can be used and still be effective. The transpoase is the most expensive part of the assay. also good to know for when assaying smaller numbers of cells, how much dilution could be made.
- new transpsosome from freshly order t7-top2 and t7-bottom when testing and thus will repeat 500 cell experiment
- Will try isolating nuclei how they did, and then performing transposition how they did too. Nuclei preparation was done with cold lysis buffer, and transposition for 30 minutes at 37C. Neither have been done with THS-seq and will be tried. 37C for 30 minutes was done before, however data looked like samples incubated at 55C for 10 minutes.
- current DNase I HS protocols and ATAC-seq paper do not incubate for lysis, thus I will not incubate for lysis as before.
- sample list
1. 500 nuclei 37C 30 rep1 2. 500 nuclei 55C rep1 3. 500 cells clysis 37C 30 rep1 4. 500 cells clysis 55C rep1 5. 2X tspsome dilution 500 cells 55C 6. 4X tspsome dilution 500 cells 55C 7. 8X tspsome dilution 500 cells 55C 8. 16X tspsome dilution 500 cells 55C 9. 32X tspsome dilution 500 cells 55C 10. 64X tspsome dilution 500 cells 55C 11. pure jurkat DNA 3 ng 12. NTC
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. make pure DNA controls for the samples if needed.
3. samples this time:
- sample list
1. 500 nuclei 37C 30 rep1 2. 500 nuclei 55C rep1 3. 500 cells clysis 37C 30 rep1 4. 500 cells clysis 55C rep1 5. 2X tspsome dilution 500 cells 55C 6. 4X tspsome dilution 500 cells 55C 7. 8X tspsome dilution 500 cells 55C 8. 16X tspsome dilution 500 cells 55C 9. 32X tspsome dilution 500 cells 55C 10. 64X tspsome dilution 500 cells 55C 11. pure jurkat DNA 3 ng 12. NTC
IVT Protocol[edit]
- If need to make more transposome, do first 2 steps. If not goto step 3.
Generation of T7tspn-top2 transposome
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
Generation of custom barcode (3' side of fragments) nextera transposome
1. annealing of ME sequence to Nextera transposon sequence
- a. Make 100 uM stock solution of (NXTA_ME_BOT, "pMENTS") and (NXTA_ME_B, "B-METS").
- 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 Nextera transposon 1.25 uL of 100% sterile glycerol 2.50 uL of Ez-TN5 transposase
- store at -20, is good for a year
3. Prepare samples, lyse cells with lysis buffer
Samples:
- sample list
1. 500 nuclei 37C 30 rep1 2. 500 nuclei 55C rep1 3. 500 cells clysis 37C 30 rep1 4. 500 cells clysis 55C rep1 5. 2X tspsome dilution 500 cells 55C 6. 4X tspsome dilution 500 cells 55C 7. 8X tspsome dilution 500 cells 55C 8. 16X tspsome dilution 500 cells 55C 9. 32X tspsome dilution 500 cells 55C 10. 64X tspsome dilution 500 cells 55C 11. pure jurkat DNA 3 ng 12. NTC
PREPARE NUCLEI (samples 1-4)
- ALL CELLS
- 1. count cells, spin down all cells at 500 g for 5 minutes
- 2. wash with cold 1X PBS, centrifuge 500 g for 5 minutes, resuspend to 500 cells/uL
NUCLEI SAMPLES
- 1. Aliquot 500 cells to 200 uL tube (~1-2 uL)
- 2. bring to 5 uL with cold 1X PBS. add 1 volume cold 2X Lysis Buffer, mix well (10 uL total)
- 3. spin down at 500 g for 10 min using refrigerated centrifuge
- 4. carefully remove most of supernatent (transposition ready)
CELL SAMPLES (6-10)
- 1. refrigerate and warm LB.
- 2. add warm or cold lysis buffer to cell aliquots, mix briefly
- 3. transposition ready
- 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.
- 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
4. transposition reaction, using (T7tspn-top2)
TRANSPOSOME SERIAL DILUTION; DILUTION BUFFER IS 1 VOLUME GLYCEROL WITH 1 VOLUME EB BUFFER
SAMPLES 1,3 37C FOR 30 MINUTES
SAMPLES 2,4-12, 55c 10 MINUTES
- 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) 1.4 uL Nuclease free water .75 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)
CLEAN WITH ZYMO RNA CLEAN AND CONCENTRATOR NOW
8. Clean RNA with AMPURE RNA beads. elute in 20 uL.
- Protocol changes:
- 1. add 30 uL to IVT reaction to bring to 50 uL total before starting protocol.
- 2. Use 1.8:1 ratio of beads, thus 80 uL of beads for 50 uL of sample in this case
- 3. Drying time after the washes is 3 minutes.
- elute in 20 uL and concentrate to 10 uL
- can quantitate with Qubit or on TBU gel.
AFTER IVT RNA HAS ALREADY BEEN GENERATED AND CLEANED WITH AMPURE BEADS
Samples this time:
Samples:
- sample list
1. 500 nuclei 37C 30 rep1 2. 500 nuclei 55C rep1 3. 500 cells clysis 37C 30 rep1 4. 500 cells clysis 55C rep1 5. pure jurkat DNA 3 ng 6. NTC
- Amounts of RNA and water to add for MMLV RT (50 ng RNA)
8. single strand synthesis MMLV RT (Clontech)
- Followed protocol for SMART MMLV Reverse Transcriptase
20 uL reaction 1. Add 2.5 uL 20 uM (100 ng total) RANDOM HEXAMERS to RNA sample. Bring to final volume of 12.5 uL with Nuclease free H2O 2. heat the mixture to 70C fo 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 mM DTT 1 uL N-H20 .5 uL SMART MMLV RT and mix (ADD LAST!!!!!) ____ 20 uL total 4. Incuvate first at Room Temperature for 10 minutes. Then incubate at 42C for 60 minutes. 5. Terminate the reaction by heating at 70C for 10 minutes
9. RNase H digestion
- Use .5 Units for 20 uL reaction. Stock is at 5 Units/ul, thus dilute 10X and use 1 uL per reaction.
a. Add 1 uL of 10X diluted Rnase H to the reaction.
b. Incubate for 20 minutes at 37C.
10. Second strand synthesis: Adding sss_NPA_prmr primer and polymerases. Can incubate at higher temps (usually at 16C since RNA is nicked) since sss_NPA_prmr has a high Tm.
a. Add 2.5 uL of sss_NPA_prmr to each reaction. b. Incubate solutions for 2 minutes at 65C. Cool immediately on ice. c. Add 2 uL of dNTPs to each reaction. d. Add polymerases etc to reactions DNAP1: Dilute 2X to 5U/uL and add 1 uL to the reaction. Incubate at 37C for 30 minutes (stock conc. 10U/uL) Klenow exo-: Add 1 uL of 5U/uL. Incubate at 37C for 30 minutes. BST 2.0: Add 2.5 uL 10X BST buffer, dilute enzyme 2X to 4 U/ul, add 1 uL to the reaction. Incubate at 65C for 30 minutes BST 2.0 No buf: dilute enzyme 2X to 4 U/ul, add 1 uL to the reaction. Incubate at 65C for 30 minutes taq2X: Add 25 uL, Incubate at 72C for 10 minutes NTC: Just add 1 uL of Klenow exo-
11. Clean DNA with with Zymo DNA Clean & Concentrator-5 Kit OR AMPURE beads depending. elute in 20 uL and concentreate . (can also use qiaquick)
USED AMPURE DNA BEADS AT 1:1.8X RATIO
can quantitate with Qubit or on TBU gel.
11.5. Eluted DNA into 20 uL per sample, since when eluting in smaller amounts more sample is lost. Thus after elution used vaccum centrifuge commentator to concentrate sample size to ~4-5 uL per sample.
- Also ~4-5 uL per sample is preferred for transposition, which has always been performed in low volume solutions. Additionally less transposome will be needed when doing that.
12. Fragmenting and 3' End tagging with Custom Nextera transposome
1 uL nextera LMW buffer 2 uL lysed/pure genomic DNA (X ng/pg DNA) X uL Nuclase free H2O .8 uL prepared T7 transposomes (MAKE SURE TO ADD LAST) (if was proportional to shendure would use .625 uL) ___________ 5 uL total solution
- Incubate at 55C for 10 minutes, cool briefly on ice after
13. 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
14. Fill in reaction
- Add 6 uL 2X taq polymerase, run at 72C for 3 minutes. (same as nextera)
CAN TRY JUST FINISHING UP WITH TAQ2X (DID THAT THIS TIME, ADDED 12.5 UL OF 1X TAQ2X TO SOLUTION AND STOPPED AT END OF 6TH CYCLE. ADDED SYBR GREEN 1X FINAL CONCENTRATION IN SOLUTION
15. PCR addition of barcodes MAKE SURE TO USE NEXTERA INDEXES!!!!!
Samples: Index 49, 1. 500 nuclei 37C 30 rep1 Index 50, 2. 500 nuclei 55C rep1 Index 51, 3. 500 cells clysis 37C 30 rep1 Index 52, 4. 500 cells clysis 55C rep1 Index 53, 5. 2X tspsome dilution 500 cells 55C Index 54, 6. 4X tspsome dilution 500 cells 55C Index 55, 7. 8X tspsome dilution 500 cells 55C Index 56, 8. 16X tspsome dilution 500 cells 55C Index 57, 9. 32X tspsome dilution 500 cells 55C Index 58, 10. 64X tspsome dilution 500 cells 55C Index 59, 11. pure jurkat DNA 3 ng Index 60, 12. NTC
KAPA SYBR FAST qPCR mix until saturation, X35 cycles 9 uL Taq2X 2 uL primers, 2 uL F, (T7-top2-PCR-iaf, OR iaf2, iaf3) 2 uL NXTA indexes PCR_R.NXTAInd[XX], SPECIFIC TO EACH SAMPLE 4 uL H2O 1 uL 25X SYBR green 12 uL DNA template (use half RT reaction) _____________ 30 uL KAPA SYBR cycles: 95C 3min, (95C for 30s, 60C for 30s, 72C for 2 min) X35, 72C for 5 min, 4C forever
- terminate before curves saturate (usually cycle 6-8)
16. Gel Size selection
- gel size select from 400-800 bp, follow gel size selection protocol
- do not need to include controls.
results[edit]
- RNA quantification results
' | ng/ul | intensity | ' | ' | ' | ' | ' | ' | ' |
5 | 1430.144755 | ||||||||
2.5 | 803.5128811 | in 10 ul | for 50 ng | H2O to add | for 100 ng | H2O to add | |||
1. 500 nuclei 37C 30 rep1 | 79.58598643 | 20125.32734 | 795.8598643 | 0.628251307 | 9.371748693 | 1.256502614 | 8.743497386 | ||
2. 500 nuclei 55C rep1 | 14.82571563 | 3892.987396 | 148.2571563 | 3.372518483 | 6.627481517 | 6.745036966 | 3.254963034 | ||
3. 500 cells clysis 37C 30 rep1 | 86.2967791 | 21807.40598 | 862.967791 | 0.5793959 | 9.4206041 | 1.1587918 | 8.8412082 | ||
4. 500 cells clysis 55C rep1 | 151.6641997 | 38191.92969 | 1516.641997 | 0.329675692 | 9.670324308 | 0.659351384 | 9.340648616 | ||
5. 2X tspsome dilution 500 cells 55C | 99.13764045 | 25026.00318 | 991.3764045 | 0.504349304 | 9.495650696 | 1.008698609 | 8.991301391 | ||
6. 4X tspsome dilution 500 cells 55C | 105.1787771 | 26540.23071 | 1051.787771 | 0.475381074 | 9.524618926 | 0.950762147 | 9.049237853 | ||
7. 8X tspsome dilution 500 cells 55C | 56.37001542 | 14306.18037 | 563.7001542 | 0.886996387 | 9.113003613 | 1.773992774 | 8.226007226 | ||
8. 16X tspsome dilution 500 cells 55C | 48.38993031 | 11948.9444 | 483.8993031 | 1.033272825 | 8.966727175 | 2.06654565 | 7.93345435 | ||
9. 32X tspsome dilution 500 cells 55C | 17.16508499 | 4943.342816 | 171.6508499 | 2.912889743 | 7.087110257 | 5.825779486 | 4.174220514 | ||
10. 64X tspsome dilution 500 cells 55C | 7.812784094 | 2845.061999 | 78.12784094 | 6.399767279 | 3.600232721 | 12.79953456 | -2.799534557 | ||
11. pure jurkat DNA 3 ng | 253.44525 | 57955.12499 | 2534.4525 | 0.197281267 | 9.802718733 | 0.394562534 | 9.605437466 | ||
12. NTC | 1.86741619 | 1511.160153 | 18.6741619 | 26.77496332 | -16.77496332 | 53.54992664 | -43.54992664 | ||
- RNA after ~16.5 hours IVT, run on TBU gel
File:ZhangLab 2 2013-11-19 13hr 29min-labeled.jpg File:ZhangLab 2 2013-11-19 13hr 32min-labeled.jpg
- qPCR curves for adding barcodes. samples are in order presented.
- used T7-top2-PCR-iaf2, and thus curves are working and better.
- Stopped at end of 10th cycle, since that is what was done for other assays. in future can see when curves saturate.
- 32X and 64X transpsome samples also went up, will see what the results are after sequencing.
Samples: Index 49, 1. 500 nuclei 37C 30 rep1 Index 50, 2. 500 nuclei 55C rep1 Index 51, 3. 500 cells clysis 37C 30 rep1 Index 52, 4. 500 cells clysis 55C rep1 Index 53, 5. 2X tspsome dilution 500 cells 55C Index 54, 6. 4X tspsome dilution 500 cells 55C Index 55, 7. 8X tspsome dilution 500 cells 55C Index 56, 8. 16X tspsome dilution 500 cells 55C Index 57, 9. 32X tspsome dilution 500 cells 55C Index 58, 10. 64X tspsome dilution 500 cells 55C Index 59, 11. pure jurkat DNA 3 ng Index 60, 12. NTC
File:2013-11-19 R13 500 cell repeat, traspsome dilution testing.bmp
- TBE gel after PCR barcode addition
File:ZhangLab 2 2013-11-20 11hr 48min-labeled.jpg File:ZhangLab 2 2013-11-20 11hr 51min-labeled.jpg
- amounts used for gel size selection
11/20/2013 | ng/ul | intensity | ' | ' |
5 | 3595.746978 | |||
2.5 | 2132.81423 | ul for 10 ng | ||
1. 500 nuclei 37C 30 rep1 | 0.999948015 | 1255.024161 | 0.999948015 | 10.00051988 |
2. 500 nuclei 55C rep1 | -0.387450647 | 443.1557864 | 1 | 10 |
3. 500 cells clysis 37C 30 rep1 | 2.065641523 | 1878.639334 | 2.065641523 | 4.841111048 |
4. 500 cells clysis 55C rep1 | 0.387333954 | 896.5388927 | 0.387333954 | 10 |
5. 2X tspsome dilution 500 cells 55C | -0.017385319 | 659.7080609 | 1 | 10 |
6. 4X tspsome dilution 500 cells 55C | 0.948604496 | 1224.979315 | 0.948604496 | 10.54180119 |
7. 8X tspsome dilution 500 cells 55C | 1.253904246 | 1403.632516 | 1.253904246 | 7.975090626 |
8. 16X tspsome dilution 500 cells 55C | 0.404996193 | 935.3681529 | 0.404996193 | 10 |
9. 32X tspsome dilution 500 cells 55C | -0.373257353 | 282.1971513 | 1 | 10 |
10. 64X tspsome dilution 500 cells 55C | 0.59298614 | 1093.143961 | 0.59298614 | 10 |
11. pure jurkat DNA 3 ng | 2.653391129 | 2822.396334 | 2.653391129 | 3.768762129 |
- gel size selction cut
File:ZhangLab 2 2013-11-20 13hr 43min-labeled.jpg
- TBE gel size selection validation
File:ZhangLab 2 2013-11-20 16hr 58min-labeled.jpg
- 1. 8X appears to be the upper limit of dilution before getting more clonal reads
- 2. 37C for 30 minutes tagmentation did better then 55C for 10 mintues
- 3. appears clonal read rate increases proportionally to the transposome dilution starting at 16X and above
- 4. not enough reads to look at peak calling differences
- 5. based on gels 8-16X is when less product is recovered
- OVERALL, USE LESS THEN 8X DILUTION OF TRANSPOSOME FOR ASSAY WITH 3 NG OF INPUT DNA
- HOWEVER APPEARS 1-2X DILUTION OF TRANSPOSOME IS STILL MOST OPTIMAL
future and conclusions[edit]
Blah