Brandon:LabNotes/Project1/2012-11-16: Difference between revisions
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GCTCGAACATTAGAGCATACGGCAGAAGACGAAC | GCTCGAACATTAGAGCATACGGCAGAAGACGAAC | ||
[[File:ZhangLab 2 2012-11-17 18hr 06min-labeled.jpg|600px]] | |||
*TBU gel after barcode/illuminia adaptor addition. TBE gel had thick bands so wanted to make sure it was still right sized fragments and not DNA hairballs. | *TBU gel after barcode/illuminia adaptor addition. TBE gel had thick bands so wanted to make sure it was still right sized fragments and not DNA hairballs. | ||
[[File:ZhangLab 2 2012-11-18 12hr 58min-labeled.jpg|600px]] | |||
*gel size selection | *gel size selection | ||
*amount of sample to combined for each sample | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''11/19/2012''' | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
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| align="center" style="background:#f0f0f0;"|'''''' | |||
|- | |||
| 5 well gel #1|||||||||||||||| | |||
|- | |||
| nextera samples||||||||||for 100 ng/sample||||1000||ug total | |||
|- | |||
| 150K||7||40.55066601||ng/ul||||2.466050742||ul|||| | |||
|- | |||
| 50K||8||48.74365792||ng/ul||||2.051548946||ul|||| | |||
|- | |||
| 10K||9||36.95263753||ng/ul||||2.706166777||ul|||| | |||
|- | |||
| 1000||10||71.16046002||ng/ul||||1.405274783||ul|||| | |||
|- | |||
| 500||11||76.33459351||ng/ul||||1.310022041||ul|||| | |||
|- | |||
| 100||12||78.83379854||ng/ul||||1.268491457||ul|||| | |||
|- | |||
| 60 ng||13||31.49869165||ng/ul||||3.174735037||ul|||| | |||
|- | |||
| 6 ng||14||20.50083472||ng/ul||||4.877850163||ul|||| | |||
|- | |||
| 3 ng||15||38.41115138||ng/ul||||2.603410635||ul|||| | |||
|- | |||
| 600 pg||16||80.47317293||ng/ul||||1.242650145||ul|||| | |||
|- | |||
| ||||||||||23.10620073|||||| | |||
|- | |||
| 5 well gel #2|||||||||||||||| | |||
|- | |||
| IVT samples||||||||||for 150 ng/sample||||1050||ug total | |||
|- | |||
| IVT 1000||1||84.69677408||ng/ul||||1.771023768||ul|||| | |||
|- | |||
| IVT 500||2||90.35440764||ng/ul||||1.660129305||ul|||| | |||
|- | |||
| IVT 100||3||73.85377967||ng/ul||||2.031040262||ul|||| | |||
|- | |||
| 6 ng||4||86.09277703||ng/ul||||1.742306442||ul|||| | |||
|- | |||
| 3 ng||5||78.83966134||ng/ul||||1.902595692||ul|||| | |||
|- | |||
| 600 pg||6||84.3488657||ng/ul||||1.778328597||ul|||| | |||
|- | |||
| IVT 1000.2||23||68.22453649||ng/ul||||2.198622486||ul|||| | |||
|- | |||
| ||||||||||13.08404655|||||| | |||
|- | |||
| | |||
|} | |||
*NEXTERA COMBINED SAMPLES | |||
[[File:ZhangLab 2 2012-11-19 12hr 29min-labeled.jpg|600px]] | |||
*IVT COMBINED SAMPLES | |||
[[File:ZhangLab 2 2012-11-19 12hr 30min-labeled.jpg|600px]] | |||
*validating gel size selection | *validating gel size selection | ||
**wierd with IVT sample. seems like there is alot of junk or who knows what. though is visible in the nextera samples some too. band not really visible in IVT samples. | |||
[[File:ZhangLab 2 2012-11-19 19hr 08min-labeled.jpg|600px]] | |||
*sutmitted samples for sequencing. nextera samples for most of flow cell, IVT samples to test and see what is wrong.. and if it is working. | |||
'''sequencing data analysis, unmappables''' | |||
*looked at unmappables in samples to see what the issue was. | |||
*turns out to be PAP is working and polyAing everything, and those are most of the viable fragments. thus seems like fragmenting of RNA is not working | |||
*index49 analysis (1000 cells IVT) | |||
*file to big to upload and is on genome minor, HL136 | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''sequences containing''' | |||
| align="center" style="background:#f0f0f0;"|'''sequence''' | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
|- | |||
| 85390||aaaaaaaaaaaaaaaaaaaaaaaaaaaaaa||30 A's | |||
|- | |||
| 210,927 ||AAAAAAAAAAAAAAAAA||(17) epic polyA | |||
|- | |||
| 11,151 ||TTCAGCAGGAATGCC||3' adaptor after T20. Thus is 5' end polyA'ed, then PCRed. | |||
|- | |||
| 101,765 ||aaaaaaaaaaaaaaaaaaaaaaaa||24 A"s | |||
|- | |||
| 531 ||TGTATAAGAGACAG||5' adaptor right hand end (few are concatmars of that sequence) | |||
|- | |||
| | |||
|} | |||
*Index52 analysis (6 ng pure DNA): | |||
*[[media:Indx52_final_analysis.xlsx]] | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''sequences containing''' | |||
| align="center" style="background:#f0f0f0;"|'''sequence''' | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
|- | |||
| 57988||aaaaaaaaaaaaaaaaaaaaaaaaaaaaaa||30 A's|||| | |||
|- | |||
| 133993||AAAAAAAAAAAAAAAAA||(17) epic polyA|||| | |||
|- | |||
| 6421||TTCAGCAGGAATGCC||3' adaptor after T20. Thus is 5' end polyA'ed, then PCRed.|||| | |||
|- | |||
| 62270||aaaaaaaaaaaaaaaaaaaaaaaa||24 A"s|||| | |||
|- | |||
| 187||TGTATAAGAGACAG||5' adaptor right hand end (few are concatmars of that sequence)|||| | |||
|- | |||
| | |||
|} | |||
Line 456: | Line 584: | ||
'''conclusions''' | '''conclusions''' | ||
*now know that IVT was defective in run on [http://genome-tech.ucsd.edu/LabNotes/index.php/Brandon:LabNotes/Project1/2012-10-26 Octover 26 (round 3)]. This will be a checkpoint after every DNA accessibility run using the transposome methods and IVT. Generation of 300+ ng of RNA is essential for the analysis to work, as seen in [http://genome-tech.ucsd.edu/LabNotes/index.php/Brandon:LabNotes/Project1/2012-8-6 august 6 run, round 2]. Then all troubeshooting is with RNA-seq after IVT to ensure the assay is working. Nextera samples did work and are being used for sequencing. | |||
' | |||
*can try more experiments increase ribonucleotide amounts for IVT. seemed to have an effect with 1000 cells, can try for lesser cell amounts that have more trouble generating RNA from IVT. Also got the most successful reads of all the samples, and 45% more than the other 1000 cell sample. | |||
*also seems that the curves rising during PCR addition of barcodes are essential for a successful library. Viable fragments are created, but not at an efficient rate. | |||
*after sequencing analysis it looks like RNA fragmentation did not work. The unmappable fragments are either short fragments that have been polyA'ed or transposon sequences (read primer and ME sequence) that have been polyA'ed, where no insert was present. IVT creates long RNA fragments, which need to be fragmented by RNase III in order to be sequenceable. | |||
*PAP and adding polyA tails to the RNA did work however. Most unmappable fragments are long polyA stretches. | |||
*FUTURE: will trouble shoot Rnase III fragmenting, and purchase new RNase III from NEB, and check fragmentation sizes with the Agilent bio analyzer in paulssons lab. Will use test RNA generated recently to test fragmentation. | |||
*compare old RNase III and new RNase III, to definitively conclude it was RNAase III issues. | |||
Latest revision as of 02:47, 4 December 2012
Custom transposon with IVT amplification, round 3 repeat of IVT samples[edit]
- will repeat IVT assay on 1000, 500, 100 cells. Nextera transposition worked fine in previous assay and will be used for sequencing.
- procotols from shendure paper, shendure paper transposition
- using T7-top2 for transposition reactions t7tspn-top2
- Improvements from last assay
- GM12878 DNA will be purified from GM12878 using RNase A (or Rnase mixture) and Protease AND proteinase K. Highly accessible regions are still highly accessible in pure DNA samples, want want to ensure proteins are not bound and RNA is not interfering. If those regions are still accessible then dunno why..
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: 1. 1000 cells IVT 2. 500 cells IVT 3. 100 cells IVT 4. 6 ng purified DNA IVT 5. 3 ng purified DNA IVT 6. 600 pg purified DNA IVT 17. 1000 cells lysed without transposome complex IVT 18. pure DNA only (6 ng) IVT 19. Nuclease free H20 only IVT 23. 1000 cells IVT (double [NT] for 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
3. Prepare samples, lyse cells with lysis buffer
use pure GM12878 DNA
Samples: 1. 1000 cells IVT 2. 500 cells IVT 3. 100 cells IVT 4. 6 ng purified DNA IVT 5. 3 ng purified DNA IVT 6. 600 pg purified DNA IVT 17. 1000 cells lysed without transposome complex IVT 18. pure DNA only (6 ng) IVT 19. 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. 500 IVT | 3. 100 IVT | 17. 1K IVT con | 19. NTC IVT | 23. 1K 2X IVT [NT] |
cells | 1 ul | 1 ul | 1 ul | 1 ul | 1 ul PBS | 1 ul |
2X LB | 1 ul | 1 ul | 1 ul | 1 ul | 1 ul | 1 ul |
4. transposition reaction, using (T7tspn-top2)
- 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
- !!!!! sample 23 used 2 uL of each NT!!!!!!!
- 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 !!!!! sample 23 used 2 uL of each NT!!!!!!! b. Incubate reactions at 37C overnight for ~16 hours. (>10 uM limiting nucleotide)
8. Clean RNA with with Zymo RNA Clean & Concentrator-5 Kit
- can quantitate with Qubit if needed, and run TBU-gel
9. Perform RNase III fragmentation (NEB):
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 95C for 2 minutes. *c. cool to RT at 0.1 C/s 2. Mix the following components in a sterile PCR tube: X uL Purified mRNA + blocking primer (50-250 nanograms) .5 uL RNase III (1 unit/μl) 1 uL RNase III Reaction Buffer (10X) 5.5 uL Nuclease-Free Water add in DNA primer to protect 5' end since don't want degradation?? _______ 10 uL total volume 3. Incubate in a preheated thermal cycler for 5 minutes at 37°C. 4. Transfer tube to ice.
10. Zymo RNA clean and concentrator cleanup.
- Resuspend in appropriate volume in nuclease free H2O (12 uL last time)
- not doing protease digestion after RNA fragmentation since seems it wasn't as effective in previous RNase III fragmentation
11. Poly(A) Addition with polyA polymerase (Enzymatics)
- enzymatics PolyA polymerase.
a. assemble reaction: 2 uL 5X SMART MMLV first strand buffer (rather than 10X polyA buffer) 1 uL polyA enzyme 1 uL 10 mM ATP bring to 10 uL with RNA or w/e b. Incubate at 37C for 10 minutes c. Heat inactivate at 70C for 20 minutes. (rui and NEB)
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 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 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. 1000 cells IVT Index 50, N2 adaptor 2. 500 cells IVT Index 51, N2 adaptor 3. 100 cells IVT Index 52, N2 adaptor 4. 6 ng purified DNA IVT Index 53, N2 adaptor 5. 3 ng purified DNA IVT Index 54, N2 adaptor 6. 600 pg purified DNA IVT Index 65, N2 adaptor 17. 1000 cells lysed without transposome complex IVT Index 66, N2 adaptor 18. pure DNA only (6 ng) IVT Index 67, N2 adaptor 19. Nuclease free H20 only IVT
KAPA SYBR FAST qPCR mix until saturation, X35 cycles 25 uL KAPA SYBR FAST qPCR mix 4 uL primers, 2 uL F, 2 uL R (T7-top2-PCR-iaf or T7-top3-PCR-iaf) and (PCR_R.N2Ind[XX]) 1 uL H2O 20 uL DNA template (use whole RT reaction) 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]
IVT amplification results/gels
- ran IVT overnight for 20 hours, 30 mins ish.
- eluted in 13 ul
- RNA amounts from gel quantification
sample | ' | ng/ul | total ng |
IVT 1000 | 1 | 123.1320964 | 1600.717253 |
IVT 500 | 2 | 65.93287018 | 857.1273123 |
IVT 100 | 3 | 24.9498017 | 324.3474221 |
6 ng | 4 | 119.2245472 | 1549.919114 |
3 ng | 5 | 18.17303159 | 236.2494107 |
600 pg | 6 | 1.815361788 | 23.59970325 |
IVT 1000.2 | 23 | 126.4626841 | 1644.014893 |
- Used 1 uL for TBU gel, 1. RNA eluted in 13 uL N-H20 total.
- graph of ng RNA versus cell number for IVT RNA amplification.
- RNA after IVT amplification, TBU gel. Expected more RNA in accessibility samples but still satisfactory total amounts.
File:ZhangLab 2 2012-11-17 12hr 22min-labeled.jpg
- qPCR curves for barcode/adaptor addition
- seems to have done the same thing as the previous assay. not sure why. checked when i did the procedure months ago and the same thing was seen in some of the samples. some samples worked. some did not. however, when doing cloning/transformation the correct type of fragments were obtained, even if barcode addition curves went down.
Samples: 1. 1000 cells IVT 2. 500 cells IVT 3. 100 cells IVT 4. 6 ng purified DNA IVT 5. 3 ng purified DNA IVT 6. 600 pg purified DNA IVT 17. 1000 cells lysed without transposome complex IVT 18. pure DNA only (6 ng) IVT 19. Nuclease free H20 only IVT 23. 1000 cells IVT (double [NT] for IVT)
- all samples
File:2012-11-17 round 3 redo IVT barcodes all.bmp
- accessibility samples
File:2012-11-17 round 3 redo IVT barcodes accessibility samples.bmp
- pure DNA samples
File:2012-11-17 round 3 redo IVT barcodes pure DNA samples.bmp
- TBE gel after barcode/illuminia adaptor addition. TBE gel, 50 uL sample total
fragments above 127 bp will have an insert, at least 157 for good insert 5’ end GGGAGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG 3’ end T20VN_PE_R AAAAAAAAAAAAAAAAAAAAACTAGCCTTCTCGCCAAGTCGTCCTTACG 3’ end of PCR_R.N2Ind[XX] with ILA adaptor GCTCGAACATTAGAGCATACGGCAGAAGACGAAC
File:ZhangLab 2 2012-11-17 18hr 06min-labeled.jpg
- TBU gel after barcode/illuminia adaptor addition. TBE gel had thick bands so wanted to make sure it was still right sized fragments and not DNA hairballs.
File:ZhangLab 2 2012-11-18 12hr 58min-labeled.jpg
- gel size selection
- amount of sample to combined for each sample
11/19/2012 | ' | ' | ' | ' | ' | ' | ' | ' |
5 well gel #1 | ||||||||
nextera samples | for 100 ng/sample | 1000 | ug total | |||||
150K | 7 | 40.55066601 | ng/ul | 2.466050742 | ul | |||
50K | 8 | 48.74365792 | ng/ul | 2.051548946 | ul | |||
10K | 9 | 36.95263753 | ng/ul | 2.706166777 | ul | |||
1000 | 10 | 71.16046002 | ng/ul | 1.405274783 | ul | |||
500 | 11 | 76.33459351 | ng/ul | 1.310022041 | ul | |||
100 | 12 | 78.83379854 | ng/ul | 1.268491457 | ul | |||
60 ng | 13 | 31.49869165 | ng/ul | 3.174735037 | ul | |||
6 ng | 14 | 20.50083472 | ng/ul | 4.877850163 | ul | |||
3 ng | 15 | 38.41115138 | ng/ul | 2.603410635 | ul | |||
600 pg | 16 | 80.47317293 | ng/ul | 1.242650145 | ul | |||
23.10620073 | ||||||||
5 well gel #2 | ||||||||
IVT samples | for 150 ng/sample | 1050 | ug total | |||||
IVT 1000 | 1 | 84.69677408 | ng/ul | 1.771023768 | ul | |||
IVT 500 | 2 | 90.35440764 | ng/ul | 1.660129305 | ul | |||
IVT 100 | 3 | 73.85377967 | ng/ul | 2.031040262 | ul | |||
6 ng | 4 | 86.09277703 | ng/ul | 1.742306442 | ul | |||
3 ng | 5 | 78.83966134 | ng/ul | 1.902595692 | ul | |||
600 pg | 6 | 84.3488657 | ng/ul | 1.778328597 | ul | |||
IVT 1000.2 | 23 | 68.22453649 | ng/ul | 2.198622486 | ul | |||
13.08404655 | ||||||||
- NEXTERA COMBINED SAMPLES
File:ZhangLab 2 2012-11-19 12hr 29min-labeled.jpg
- IVT COMBINED SAMPLES
File:ZhangLab 2 2012-11-19 12hr 30min-labeled.jpg
- validating gel size selection
- wierd with IVT sample. seems like there is alot of junk or who knows what. though is visible in the nextera samples some too. band not really visible in IVT samples.
File:ZhangLab 2 2012-11-19 19hr 08min-labeled.jpg
- sutmitted samples for sequencing. nextera samples for most of flow cell, IVT samples to test and see what is wrong.. and if it is working.
sequencing data analysis, unmappables
- looked at unmappables in samples to see what the issue was.
- turns out to be PAP is working and polyAing everything, and those are most of the viable fragments. thus seems like fragmenting of RNA is not working
- index49 analysis (1000 cells IVT)
- file to big to upload and is on genome minor, HL136
sequences containing | sequence | ' |
85390 | aaaaaaaaaaaaaaaaaaaaaaaaaaaaaa | 30 A's |
210,927 | AAAAAAAAAAAAAAAAA | (17) epic polyA |
11,151 | TTCAGCAGGAATGCC | 3' adaptor after T20. Thus is 5' end polyA'ed, then PCRed. |
101,765 | aaaaaaaaaaaaaaaaaaaaaaaa | 24 A"s |
531 | TGTATAAGAGACAG | 5' adaptor right hand end (few are concatmars of that sequence) |
- Index52 analysis (6 ng pure DNA):
- media:Indx52_final_analysis.xlsx
sequences containing | sequence | ' | ' | ' |
57988 | aaaaaaaaaaaaaaaaaaaaaaaaaaaaaa | 30 A's | ||
133993 | AAAAAAAAAAAAAAAAA | (17) epic polyA | ||
6421 | TTCAGCAGGAATGCC | 3' adaptor after T20. Thus is 5' end polyA'ed, then PCRed. | ||
62270 | aaaaaaaaaaaaaaaaaaaaaaaa | 24 A"s | ||
187 | TGTATAAGAGACAG | 5' adaptor right hand end (few are concatmars of that sequence) | ||
conclusions
- now know that IVT was defective in run on Octover 26 (round 3). This will be a checkpoint after every DNA accessibility run using the transposome methods and IVT. Generation of 300+ ng of RNA is essential for the analysis to work, as seen in august 6 run, round 2. Then all troubeshooting is with RNA-seq after IVT to ensure the assay is working. Nextera samples did work and are being used for sequencing.
- can try more experiments increase ribonucleotide amounts for IVT. seemed to have an effect with 1000 cells, can try for lesser cell amounts that have more trouble generating RNA from IVT. Also got the most successful reads of all the samples, and 45% more than the other 1000 cell sample.
- also seems that the curves rising during PCR addition of barcodes are essential for a successful library. Viable fragments are created, but not at an efficient rate.
- after sequencing analysis it looks like RNA fragmentation did not work. The unmappable fragments are either short fragments that have been polyA'ed or transposon sequences (read primer and ME sequence) that have been polyA'ed, where no insert was present. IVT creates long RNA fragments, which need to be fragmented by RNase III in order to be sequenceable.
- PAP and adding polyA tails to the RNA did work however. Most unmappable fragments are long polyA stretches.
- FUTURE: will trouble shoot Rnase III fragmenting, and purchase new RNase III from NEB, and check fragmentation sizes with the Agilent bio analyzer in paulssons lab. Will use test RNA generated recently to test fragmentation.
- compare old RNase III and new RNase III, to definitively conclude it was RNAase III issues.
Notes ETC[edit]
- since previous frag block was designed for T7-top3.
T7-frag-block-top2 5'- CTGTCTCTTATACACATCTCTGATGGCGCGAGGGAGGATCTCCC/3InvdT/ Tm= 81.45
- Effeciency of DNase I to degrade ssDNA and DNA:RNA hybrids is 500X less effecient, according to Invitrogen site thus use longer incubation?
Barcodes used:
Samples: Index 49, N2 adaptor 1. 1000 cells IVT Index 50, N2 adaptor 2. 500 cells IVT Index 51, N2 adaptor 3. 100 cells IVT Index 52, N2 adaptor 4. 6 ng purified DNA IVT Index 53, N2 adaptor 5. 3 ng purified DNA IVT Index 54, N2 adaptor 6. 600 pg purified DNA IVT Index 55, NX adaptor 7. 150K cells nextera tagmentation Index 56, NX adaptor 8. 50K cells nextera tagmentation Index 57, NX adaptor 9. 10K cells nextera tagmentation Index 58, NX adaptor 10. 1000 cells nextera tagmentation Index 59, NX adaptor 11. 500 cells nextera tagmentation Index 60, NX adaptor 12. 100 cells nextera tagmentation Index 61, NX adaptor 13. 60 ng purified DNA nextera tagmentation Index 62, NX adaptor 14. 6 ng purified DNA nextera tagmentation Index 63, NX adaptor 15. 3 ng purified DNA nextera tagmentation Index 64, NX adaptor 16. 600 pg purified DNA nextera tagmentation Index 65, N2 adaptor 17. 1000 cells lysed without transposome complex IVT Index 66, N2 adaptor 18. pure DNA only (6 ng) IVT Index 67, N2 adaptor 19. Nuclease free H20 only IVT Index 68, NX adaptor 20. 1000 cells lysed without transposome complex nextera tagmentation Index 69, NX adaptor 21. pure DNA only (6 ng) nextera tagmentation Index 70, NX adaptor 22. Nuclease free H20 only nextera tagmentation
- N2 adaptor with illuminia adatpor on the 5' end.
My modifications for T7-tspns for 5' addition primers To use with (T7-top and T7-top2) transposons 5’- [AATGATACGGCGACCACCGA][GATCT][CTCCCTCGCGCCATCAGAGAT] -3’ (Tm=86.59) ILA adaptor blue Top2-5’end (T7-top2-PCR-iaf) Tm=66.79 (can be used if top1 is used for transposition)
- N2 adaptors with barcodes and illuminia adaptors (N9_PE_R and T20VN_PE_R)
Barcode ID | Barcode | Primer | Primer Name |
Ind49 | ACACAG | CAAGCAGAAGACGGCATACGAGATACACAGCTCGGCATTCCTGCTGAACCGCTCTT | PCR_R.N2Ind49 |
Ind50 | AAAGGT | CAAGCAGAAGACGGCATACGAGATAAAGGTCTCGGCATTCCTGCTGAACCGCTCTT | PCR_R.N2Ind50 |
Ind51 | GCGATA | CAAGCAGAAGACGGCATACGAGATGCGATACTCGGCATTCCTGCTGAACCGCTCTT | PCR_R.N2Ind51 |
Ind52 | CGTGTC | CAAGCAGAAGACGGCATACGAGATCGTGTCCTCGGCATTCCTGCTGAACCGCTCTT | PCR_R.N2Ind52 |
Ind53 | GTAGAA | CAAGCAGAAGACGGCATACGAGATGTAGAACTCGGCATTCCTGCTGAACCGCTCTT | PCR_R.N2Ind53 |
Ind54 | GGACGT | CAAGCAGAAGACGGCATACGAGATGGACGTCTCGGCATTCCTGCTGAACCGCTCTT | PCR_R.N2Ind54 |
Ind55 | AGTCGA | CAAGCAGAAGACGGCATACGAGATAGTCGACTCGGCATTCCTGCTGAACCGCTCTT | PCR_R.N2Ind55 |
Ind56 | GTCTGA | CAAGCAGAAGACGGCATACGAGATGTCTGACTCGGCATTCCTGCTGAACCGCTCTT | PCR_R.N2Ind56 |
Ind57 | GAAGGA | CAAGCAGAAGACGGCATACGAGATGAAGGACTCGGCATTCCTGCTGAACCGCTCTT | PCR_R.N2Ind57 |
Ind58 | ATGCTG | CAAGCAGAAGACGGCATACGAGATATGCTGCTCGGCATTCCTGCTGAACCGCTCTT | PCR_R.N2Ind58 |
Ind59 | TCTATC | CAAGCAGAAGACGGCATACGAGATTCTATCCTCGGCATTCCTGCTGAACCGCTCTT | PCR_R.N2Ind59 |
Ind60 | ATCTGT | CAAGCAGAAGACGGCATACGAGATATCTGTCTCGGCATTCCTGCTGAACCGCTCTT | PCR_R.N2Ind60 |
Ind61 | ATAGAG | CAAGCAGAAGACGGCATACGAGATATAGAGCTCGGCATTCCTGCTGAACCGCTCTT | PCR_R.N2Ind61 |
Ind62 | GCTAAA | CAAGCAGAAGACGGCATACGAGATGCTAAACTCGGCATTCCTGCTGAACCGCTCTT | PCR_R.N2Ind62 |
Ind63 | ACCAGG | CAAGCAGAAGACGGCATACGAGATACCAGGCTCGGCATTCCTGCTGAACCGCTCTT | PCR_R.N2Ind63 |
Ind64 | CCAACT | CAAGCAGAAGACGGCATACGAGATCCAACTCTCGGCATTCCTGCTGAACCGCTCTT | PCR_R.N2Ind64 |
Ind65 | AAGGAA | CAAGCAGAAGACGGCATACGAGATAAGGAACTCGGCATTCCTGCTGAACCGCTCTT | PCR_R.N2Ind65 |
Ind66 | CCTCCA | CAAGCAGAAGACGGCATACGAGATCCTCCACTCGGCATTCCTGCTGAACCGCTCTT | PCR_R.N2Ind66 |
Ind67 | CACGTC | CAAGCAGAAGACGGCATACGAGATCACGTCCTCGGCATTCCTGCTGAACCGCTCTT | PCR_R.N2Ind67 |
Ind68 | CATAAC | CAAGCAGAAGACGGCATACGAGATCATAACCTCGGCATTCCTGCTGAACCGCTCTT | PCR_R.N2Ind68 |
Ind69 | CCATAT | CAAGCAGAAGACGGCATACGAGATCCATATCTCGGCATTCCTGCTGAACCGCTCTT | PCR_R.N2Ind69 |
Ind70 | GAAGTC | CAAGCAGAAGACGGCATACGAGATGAAGTCCTCGGCATTCCTGCTGAACCGCTCTT | PCR_R.N2Ind70 |
Ind71 | CAAAGA | CAAGCAGAAGACGGCATACGAGATCAAAGACTCGGCATTCCTGCTGAACCGCTCTT | PCR_R.N2Ind71 |
Ind72 | TGGCAG | CAAGCAGAAGACGGCATACGAGATTGGCAGCTCGGCATTCCTGCTGAACCGCTCTT | PCR_R.N2Ind72 |
- nextera Adaptor 1, matches blue primer, CCTCCCTCGCGCCATCAG, 18nt
Adaptor 1*: 5′-AATGATACGGCGACCACCGAGATCTACACGCCTCCCTCGCGCCATCAG-3′
- nextera barcoded adaptor 2's and illuminia adaptors. matches orange primer, Orange primer CCTTGCCAGCCCGCTCAG, 18nt
Barcode ID | Barcode | Primer |
Ind49 | ACACAG | CAAGCAGAAGACGGCATACGAGATACACAGCGGTCTGCCTTGCCAGCCCGCTCAG |
Ind50 | AAAGGT | CAAGCAGAAGACGGCATACGAGATAAAGGTCGGTCTGCCTTGCCAGCCCGCTCAG |
Ind51 | GCGATA | CAAGCAGAAGACGGCATACGAGATGCGATACGGTCTGCCTTGCCAGCCCGCTCAG |
Ind52 | CGTGTC | CAAGCAGAAGACGGCATACGAGATCGTGTCCGGTCTGCCTTGCCAGCCCGCTCAG |
Ind53 | GTAGAA | CAAGCAGAAGACGGCATACGAGATGTAGAACGGTCTGCCTTGCCAGCCCGCTCAG |
Ind54 | GGACGT | CAAGCAGAAGACGGCATACGAGATGGACGTCGGTCTGCCTTGCCAGCCCGCTCAG |
Ind55 | AGTCGA | CAAGCAGAAGACGGCATACGAGATAGTCGACGGTCTGCCTTGCCAGCCCGCTCAG |
Ind56 | GTCTGA | CAAGCAGAAGACGGCATACGAGATGTCTGACGGTCTGCCTTGCCAGCCCGCTCAG |
Ind57 | GAAGGA | CAAGCAGAAGACGGCATACGAGATGAAGGACGGTCTGCCTTGCCAGCCCGCTCAG |
Ind58 | ATGCTG | CAAGCAGAAGACGGCATACGAGATATGCTGCGGTCTGCCTTGCCAGCCCGCTCAG |
Ind59 | TCTATC | CAAGCAGAAGACGGCATACGAGATTCTATCCGGTCTGCCTTGCCAGCCCGCTCAG |
Ind60 | ATCTGT | CAAGCAGAAGACGGCATACGAGATATCTGTCGGTCTGCCTTGCCAGCCCGCTCAG |
Ind61 | ATAGAG | CAAGCAGAAGACGGCATACGAGATATAGAGCGGTCTGCCTTGCCAGCCCGCTCAG |
Ind62 | GCTAAA | CAAGCAGAAGACGGCATACGAGATGCTAAACGGTCTGCCTTGCCAGCCCGCTCAG |
Ind63 | ACCAGG | CAAGCAGAAGACGGCATACGAGATACCAGGCGGTCTGCCTTGCCAGCCCGCTCAG |
Ind64 | CCAACT | CAAGCAGAAGACGGCATACGAGATCCAACTCGGTCTGCCTTGCCAGCCCGCTCAG |
Ind65 | AAGGAA | CAAGCAGAAGACGGCATACGAGATAAGGAACGGTCTGCCTTGCCAGCCCGCTCAG |
Ind66 | CCTCCA | CAAGCAGAAGACGGCATACGAGATCCTCCACGGTCTGCCTTGCCAGCCCGCTCAG |
Ind67 | CACGTC | CAAGCAGAAGACGGCATACGAGATCACGTCCGGTCTGCCTTGCCAGCCCGCTCAG |
Ind68 | CATAAC | CAAGCAGAAGACGGCATACGAGATCATAACCGGTCTGCCTTGCCAGCCCGCTCAG |
Ind69 | CCATAT | CAAGCAGAAGACGGCATACGAGATCCATATCGGTCTGCCTTGCCAGCCCGCTCAG |
Ind70 | GAAGTC | CAAGCAGAAGACGGCATACGAGATGAAGTCCGGTCTGCCTTGCCAGCCCGCTCAG |
Ind71 | CAAAGA | CAAGCAGAAGACGGCATACGAGATCAAAGACGGTCTGCCTTGCCAGCCCGCTCAG |
Ind72 | TGGCAG | CAAGCAGAAGACGGCATACGAGATTGGCAGCGGTCTGCCTTGCCAGCCCGCTCAG |
Read primers used for sequencing:
T7tspn-Read1 and Nextera Read 1 primer are exactly the same except for first nucleotide on 5' end. for IVT generated samples: T7tspn-Read1 5'- TCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG -3' N2RevSeq2 (read primer 2) 5'- CTCGGCATTCCTGCTGAACCGCTCTTCCGATCT -3' N2IndSeq 5'- AAGAGCGGTTCAGCAGGAATGCCGAG -3' for Nextera generated samples: Nextera Read 1 Primer: 5′- GCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG -3′ Nextera Read 2 primer: 5′- GCCTTGCCAGCCCGCTCAGAGATGTGTATAAGAGACAG -3′ Nextera Index Read Primer: 5′-CTGTCTCTTATACACATCTCTGAGCGGGCTGGCAAGGCAGACCG-3′
Stuff used for RNA-seq for BENG160 class
5’ end addition primers TSO_N10_BC[XX] [AAGCAGTGGTATCAACGCAGAGdUdU]NNNNNNNNNNTTTAGGrGrGrG adatpor1 5'- AAG CAG TGG TAT CAA CGC AGA G/ideoxyU//ideoxyU/ NNN NNN NNN NTT TAG GrGrGrG -3' P1-STRT 5’- [AATGATACGGCGACCACCGA][GATCT][AAGCAGTGGTATCAACGCAGAGT] -3’ (Tm=82.59) ILA adaptor blue Tm=64.31 adaptor1 Tm=61.02 STRT-SEQ 5'- [GATCT][AAGCAGTGGTATCAACGCAGAGTT] -3' adaptor1
3’ end addition primers T20VN_PE_R 5'-Bio-[GCATTCCTGCTGAACCGCTCTT]CCGATCTTTTTTTTTTTTTTTTTTTTTVN -3’ (Tm=78.92) adaptor2 Tm=65.68 PCR_R.N2Ind[XX] (XX= index number) 5’- [CAAGCAGAAGACGGCATACGAGAT][TACAAG]CTCG][GCATTCCTGCTGAACCGCTCTT] -3’ (Tm=87.12) ILA adaptor orange bc adaptor2 Tm=65.68
My modifications for T7-tspns for 5' addition primers To use with (T7-top and T7-top2) transposons 5’- [AATGATACGGCGACCACCGA][GATCT][CTCCCTCGCGCCATCAGAGAT] -3’ (Tm=86.59) ILA adaptor blue Top2-5’end (T7-top2-PCR-iaf) Tm=66.79 (can be used if top1 is used for transposition) To use with (T7-top3) transposon 5’- [AATGATACGGCGACCACCGA][GATCT][GGGAGACATTAAGATGTGTATAAGAGACAG] -3’ (Tm=81.40) ILA adaptor blue Top3-5’end (T7-top3-PCR-iaf) Tm=60.71