Editing
Brandon:LabNotes/Project1/2012-11-16
(section)
Jump to navigation
Jump to search
Warning:
You are not logged in. Your IP address will be publicly visible if you make any edits. If you
log in
or
create an account
, your edits will be attributed to your username, along with other benefits.
Anti-spam check. Do
not
fill this in!
===Results=== '''IVT amplification results/gels''' *ran IVT overnight for 20 hours, 30 mins ish. *eluted in 13 ul *RNA amounts from gel quantification {| {{table}} | align="center" style="background:#f0f0f0;"|'''sample''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''ng/ul''' | align="center" style="background:#f0f0f0;"|'''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. [[File:11-16 rna-quant.png|600px]] *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|600px]] *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|600px]] *accessibility samples [[File:2012-11-17 round 3 redo IVT barcodes accessibility samples.bmp|600px]] *pure DNA samples [[File:2012-11-17 round 3 redo IVT barcodes pure DNA samples.bmp|600px]] *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 5β end GGGAGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG 3β end T20VN_PE_R AAAAAAAAAAAAAAAAAAAAACTAGCCTTCTCGCCAAGTCGTCCTTACG 3β end of PCR_R.N2Ind[XX] with ILA adaptor 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. [[File:ZhangLab 2 2012-11-18 12hr 58min-labeled.jpg|600px]] *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;"|'''''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''''' | 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 **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.xlsxβ]] {| {{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)|||| |- | |} '''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.
Summary:
Please note that all contributions to ZhangLabWiki may be edited, altered, or removed by other contributors. If you do not want your writing to be edited mercilessly, then do not submit it here.
You are also promising us that you wrote this yourself, or copied it from a public domain or similar free resource (see
ZhangLabWiki:Copyrights
for details).
Do not submit copyrighted work without permission!
Cancel
Editing help
(opens in new window)
Navigation menu
Personal tools
Not logged in
Talk
Contributions
Create account
Log in
Namespaces
Page
Discussion
English
Views
Read
Edit
View history
More
Search
Navigation
Main Page
Current events
Recent changes
Random page
Investigators
Matt Cai
Song Chen
Eric Chu
Dinh Diep
Elizabeth Duong
Shicheng Guo
Alan Fung
Daniel Jacobsen
Blue Lake
Huy Lam
Alice Li
Andrew Richards
Brandon Sos
Chris Wei
Yan Wu
Kun Zhang
Tools
What links here
Related changes
Special pages
Page information