Brandon:LabNotes/Project1/2012-6-29
Measuring tagmentation efficiency cells versus pure DNA[edit]
- procotols from shendure paper, shendure paper transposition
- using T7-top2 for transposition reactions t7tspn-top2
- goal is to compare efficiency of DNA accessibility across different cellular concentrations. Also to test efficiency of tagmentation with custom transposon/transposome on pure DNA, and efficiency of amplification from IVT.
- Using GM12878 cells.
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
3. Prepare samples, lyse cells with lysis buffer
Samples: 1. 6 ng 218HFF purified DNA 2. 600 pg 218HFF purified DNA 3. 60 pg 218HFF purified DNA 4. 10,000 cells 5. 1,000 cells 6. 100 cells 7. 1000 cells without transposome complex 8. pure DNA only (6 ng) 9. 1000 cells lysed DNA only 10. Nuclease free H20 only (cells) 11. Nuclease free H20 only (pure DNA)
- 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 and MEFs) that contain 10000, 1000, 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
' | 4. 10000 | 5. 1000 | 6. 100 | 7. 1000C | 9. 1000C | 10. NTC | 11. NTC | |
cells | 1ul | 1ul | 1ul | 1ul | 1ul | 1ul PBS | 1ul PBS | |
2X LB | 1ul | 1ul | 1ul | 1ul | 1ul | 1ul | 1ul | |
- incubate at 37C for 30 mins.
4. transposition reaction, using (T7tspn-top2)
- add the below into one tube and incubate for 8 minutes at 55C.
1 uL nextera HMW buffer 2 uL lysed/pure genomic DNA (X ng/pg DNA) 1 uL Nuclease free water 1 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)
- IVT on filled in reaction and not filled in reaction in to show fill-in is working.
7. IMPORTANT!!
- samples 1-3, 11 not included in IVT
- use samples 4-10 for IVT
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) c. Clean RNA with with Zymo RNA Clean & Concentrator-5 Kit
- Now can use nanodrop or qu-bit for quantitation or run gel.
8. binding of random nonamer and MMLV RT (Clontech)
- Followed protocol for SMART MMLV Reverse Transcriptase (except with random nonamers) WITH MODIFICATIONS!!!
- higher denaturing temperature since RNA-RNA hybrids
- incubation times for random nonamers
- used 100 ng RNA for each reaction.
for 20 uL reaction a. use N9_PE_R, mix 2.5 uL of 20 uM N9_PE_R with RNA sample. Bring to final volume of 11.5 with Nuclease free H2O Mix well!! b. Heat at 85C for 3 minutes. Immediately cool on ice. c. Add the following to the reaction. (mix before adding MMLV) 4 uL 5X first strand buffer 2 uL dNTP mix 2 uL 100 uM DTT .5 uL SMART MMLV RT and mix (ADD LAST!!!!!) _______ 20 uL total d. Incuvate at 16C for 10 minutes, then 42C for 60 minutes. e. Terminate the reaction by heating at 70C for 10 minutes.
9. IMPORTANT!!!
- include all samples now!! (1-11)
second strand synthesis (qPCR) (KAPA)
KAPA SYBR FAST qPCR mix X35 cycles 25 uL KAPA SYBR 4 uL primers, 1 uL F, 1 uL R (T7-top2-PCR-iaf or T7-top3-PCR-iaf) and (PCR_R.N2Ind[XX] (23,24)) 1 uL H2O 20 uL DNA template (use whole RT reaction) KAPA SYBR cycles: 98C 3min, (98C for 30s, 60C for 30s, 72C for 1 min) X35, 72C for 5 min, 4C forever
- terminate when curves saturate
10.qiaquick cleanup or beads purification
- run on gel or w/e
- run qiaquick to clean sample before performing qPCR.
- 6 enzymes from 6 different reactions in there already.
Results[edit]
- used GM12878 lymphocytes
- used .8 uL of transposon to save on transposome mixture. according to shendure protocol, .626 uL would be the equivalent of what they used for their assay.
- used Qubit for RNA quantitation.
- samples gave roughly the same amount of RNA regradless of starting cell amounts.
sample# | description | RNA | total in sample |
4 | 10000 Cells | 33.4 ng/ul | 400.8 ng |
5 | 1000 Cells | 46.8 ng/ul | 561.6 ng |
6 | 100 Cells | 39.9 ng/ul | 478.8 ng |
7 | 1000 cells control | 2.75 ng/ul | 33 ng |
8 | pure DNA only | Out Of Range | Out Of Range |
9 | 1000 Cells only | 3.1 ng/ul | 37.2 ng |
10 | N-H2O | Out Of Range | Out Of Range |
- used random nonamer for MMLV RT for samples 4-10. used 100 ng total for RNA, except for controls where used 2.5 uL of each.
- for qPCR, used different primers for different samples
samples 4-10 T7-top2-PCR-iaf PCR_R.N2Ind[24] samples 1-3,11 T7-top2-PCR-iaf N9_PE_R
- curves were terminated at end of cycle 7 for samples 5,6,7 (10000,1000,100 cells respectively) and end of cycle 10 for the rest of the samples.
- samples 1-3 are pure DNA, and 4 is sample 11 (N-H2O). samples 5-11 are samples 4-10 in original list.
File:2012-07-02 cells testing IVT, controls, all.bmp
- ran all samples on gel to see smear.
- only samples 4,5,6 (10000,1000,100 cells respectively) have a smear. expect to see the same smear since the same amount of input RNA for each sample was used in MMLV RT.
File:ZhangLab 2 2012-07-02 19hr 03min-labeled.jpg
update 7/6/2012 IVT on pure DNA samples/IVT again on cells[edit]
Procedure:
- 1. IVT over nite on samples 1-3,11
- 2. Zymo cleaning of RNA
- 3. qubit RNA quanitaton on the samples
- 4. Random nonamer MMLV on samples 1-7,11
- 5. PCR addition of adaptors on samples 1-7,11
- primers used
T7-top2-PCR-iaf N9_PE_R barcodes 49-56
- barcodes used:
sample | barcode |
1 | 49 |
2 | 50 |
3 | 51 |
4 | 52 |
5 | 53 |
6 | 54 |
7 | 55 |
11 | 56 |
PCR information (only half of MMLV reaction used)
10 uL sample 1 uL T7-top2-PCR-iaf 1 uL barcode .5 uL N-H2O 12.5 uL KAPA SYBR fast KAPA SYBR cycles: 98C 3min, (98C for 30s, 60C for 30s, 72C for 1 min) X35, 72C for 5 min, 4C forever
- terminate when curves saturate, (was at 8 cycles)
- qubit qunitation results, including results from 6/29
7/6/2012 | ' | ' | ' | ' | ' | ' |
sample# | description | RNA | total in sample | for 200 ng | water | |
1 | 6 ng pure DNA | 37.3 ng/ul | 447.6 ng | 4.468275246 | 4.531724754 | |
2 | 600 pg pure DNA | 26.6 ng/ul | 319.2 ng | 6.26566416 | 2.73433584 | |
3 | 60 pg pure DNA | 10.9 ng/ul | 130.8 ng | 9 | 0 | |
11 | N-free H2O | Out Of Range | Out Of Range | #VALUE! | #VALUE! | |
9 | ||||||
9 | ||||||
9 | ||||||
6/29/2012 | 9 | |||||
sample# | description | RNA | total in sample | 5.988023952 | 3.011976048 | |
4 | 10000 Cells | 33.4 ng/ul | 400.8 ng | 9 | ||
5 | 1000 Cells | 46.8 ng/ul | 561.6 ng | 4.273504274 | 4.726495726 | |
6 | 100 Cells | 39.9 ng/ul | 478.8 ng | 5.012531328 | 3.987468672 | |
7 | 1000 cells control | 2.75 ng/ul | 33 ng | 72.72727273 | -63.72727273 | |
8 | pure DNA only | Out Of Range | Out Of Range | #VALUE! | #VALUE! | |
9 | 1000 Cells only | 3.1 ng/ul | 37.2 ng | 64.51612903 | -55.51612903 | |
10 | N-H2O | Out Of Range | Out Of Range | #VALUE! | #VALUE! | |
- qubit quanitation graph
- seems like with the cells there were just more cells than I thought used in the assay. Pure DNA curve looks fine with decreasing concentration.
File:2012-07-05 RNA quant graph both.png
- qPCR curves, all worked as expected.
File:2012-07-06 qPCR transposition vs. cells after IVT-labeled.bmp
- TBE gel of samples, pure DNA samples worked as expected.
File:ZhangLab 2 2012-07-06 16hr 53min-labeled.jpg
- next
- combine samples since now barcoded, do size selection and transformation cloning to make sure have right inserts. If correct then can submit for sequencing.
- 7/9/2012 update
- comibed 8.5 uL of each sample for gel size selection, since has already been barcoded. size selected for about 400 bp and about 800 bp.
File:ZhangLab 2 2012-07-07 16hr 22min-labeled.jpg
- did cloning and transformation.
- used 3 uL sample for ligation, 3 uL of ligation mixture for cloning.
used taq2x for amplification, X29 cycles for colony PCR.
12.5 uL taq2X 2 uL primers, 1 uL F, 1 uL R 5.5 uL H2O 5 uL DNA template taq2x cycles: 95C 5min, (95C for 30s, 60C for 30s, 72C for 1 min) X35, 72C for 5 min, 4C forever
- got a couple inserts.. maybe needed to stop reaction earlier for PCR? (still have fss sample left)
- sent off for sequencing, samples 1,2,4,5,10,20, 24, 27, 30, 34, 41, 43, 55, 62, 72
after sequencing analysis:
- sequence of clones file
Media:2012-07-10 custom trpn sequecing results analysis.docx
- clones stats
' | clones | % of total |
possible | 13 | 0.168831169 |
have genomic insert | 8 | 0.103896104 |
total clones | 77 | |
- Statistics based on sample
transformation | ' | samples | validated insert |
3 | ~800 bp gel cut-1 | 1-11 | 2 |
4 | ~800 bp gel cut-2 | 12-22 | 1 |
3 | ~800 bp gel cut-1 | 23-32 | |
4 | ~800 bp gel cut-2 | 34-44 | 2 |
1-1 | ~400 bp gel cut-1 | 45-55 | 1 |
2-1 | ~400 bp gel cut-2 | 56-66 | 1 |
2-1LT2 | ~400 bp gel cut-2-1 | 67-72 | 1 |
1-1LT2 | ~400 bp gel cut-1-1 | 73-77 | |
total | 8 | ||
File:ZhangLab 2 2012-07-09 13hr 21min-labeled.jpg File:ZhangLab 2 2012-07-09 13hr 22min-labeled.jpg File:ZhangLab 2 2012-07-10 12hr 04min-labeled.jpg File:ZhangLab 2 2012-07-10 12hr 06min-labeled.jpg File:ZhangLab 2 2012-07-10 14hr 30min-labeled.jpg File:ZhangLab 2 2012-07-10 14hr 31min-labeled.jpg File:ZhangLab 2 2012-07-10 14hr 38min-labeled.jpg
- ran TBU gel to make sure long double stranded products were being obtained still after PCR amplification and not balls or short DNAs.
File:ZhangLab 2 2012-07-09 16hr 37min-labeled.jpg
conclusions[edit]
- During Qubit analysis, rougly the same amount of RNA was obtained from each sample after overnight IVT. unsure why, and the 10,000 cell sample gave the least amount but only by 100 nanograms.
- It could be the amount of transposition occuring was the limiting factor.. use more transposon to fragment DNA?
- Or IVT reaction could have saturated or used up limiting nucleotide/reagent by the time it has synthesized that much RNA.
- pure DNA, and nuclease free water were out of range, and 1000 cell controls had a minimal amount of RNA which figures since cells have RNA in them.
- Random nonamer binding did not work for samples where only transposition took place, either that or not enough DNA. can't see smears when used 5 ng/ul or DNA per sample when testing for tranposition with TBU gel.
future[edit]
- sequencing result from above data and experiment are on sequencing data anlysis, tagmentation/IVT
- half of the pure DNA samples are left, will use the rest for IVT amplification, then clean with Zymo RNA cleaning kit, quant with qubit, then MMLV RT, and PCR with adaptors with different index numbers.
- use rest of IVT RNA from tagmentation on cells and do MMLV RT, PCR with adaptors with different index numbers.
- use ~200 ng or the rest of the IVT RNA for MMLV RT to maximize input coverage.
- after barcode samples, then can do size selection and cloning/transformation to show that correct inserts are being obtained. can also combine all the samples since they will all have different barcodes.
- can then submit for sequencing. Can see if pure DNA library is randomly distributed while libraries from cells center over accessible regions. (can compare to UCSC genome browser GM12878 line again).