Rui Liu: Difference between revisions
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>RuiLiu |
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==Sample record for sequencing== | ==Sample record for sequencing== | ||
7.25.11: '''RL-RNAseq-1_4-July25''' | |||
{| {{table}} border=1 | |||
| align="center" style="background:#f0f0f0;"|'''Sample''' | |||
| align="center" style="background:#f0f0f0;"|'''Original volume''' | |||
| align="center" style="background:#f0f0f0;"|'''Mappable reads''' | |||
| align="center" style="background:#f0f0f0;"|'''Modified volume''' | |||
| align="center" style="background:#f0f0f0;"|'''IndX''' | |||
|- | |||
| GFP+||2.55||0.8M||5.1||PCR_R.N2Ind1 | |||
|- | |||
| GFP-||2||1.6M||2||PCR_R.N2Ind2 | |||
|- | |||
| E9.5||3.04||1.5M||3.3||PCR_R.N2Ind3 | |||
|- | |||
| E11.5||2.39||1.2M||3.18||PCR_R.N2Ind4 | |||
|} | |||
7.13.11: '''RL-RNAseq-1_6-July12''' | 7.13.11: '''RL-RNAseq-1_6-July12''' |
Revision as of 17:49, 25 July 2011
Lab Projects
Sample record for sequencing
7.25.11: RL-RNAseq-1_4-July25
Sample | Original volume | Mappable reads | Modified volume | IndX |
GFP+ | 2.55 | 0.8M | 5.1 | PCR_R.N2Ind1 |
GFP- | 2 | 1.6M | 2 | PCR_R.N2Ind2 |
E9.5 | 3.04 | 1.5M | 3.3 | PCR_R.N2Ind3 |
E11.5 | 2.39 | 1.2M | 3.18 | PCR_R.N2Ind4 |
7.13.11: RL-RNAseq-1_6-July12
Sample ID | Source | Genome | IndX |
GFP+ | Xu Yang lab | human | PCR_R.N2Ind1 |
GFP- | Xu Yang lab | human | PCR_R.N2Ind2 |
E9.5 | Yi Zhang lab | mouse | PCR_R.N2Ind3 |
E11.5 | Yi Zhang lab | mouse | PCR_R.N2Ind4 |
E13.5m | Yi Zhang lab | mouse | PCR_R.N2Ind5 |
E13.5f | Yi Zhang lab | mouse | PCR_R.N2Ind6 |
Sample description: [1] Methods: GFP+ and GFP-: [2] E9.5 to E13.5f: [3] Shearing: [4] RNA library: [5] Final check: [6]
6.8.11: RL-Hap_RNAseq-1_13-Jun6
Sample ID | Source | Genome | IndX |
Hap1 | GM12878 | human | Nextera ID1 |
Hap2 | GM12878 | human | Nextera ID2 |
Hap3 | GM12878 | human | Nextera ID3 |
Hap4 | GM12878 | human | Nextera ID4 |
Hap5 | GM12878 | human | Nextera ID5 |
Hap6 | GM12878 | human | Nextera ID6 |
Hap7 | GM12878 | human | Nextera ID7 |
Hap8 | GM12878 | human | Nextera ID8 |
Hap9 | GM12878 | human | Nextera ID9 |
Hap10 | GM12878 | human | Nextera ID10 |
Hap11 | GM12878 | human | Nextera ID11 |
Hap12 | GM12878 | human | Nextera ID12 |
RNA-EL | Mm ES (50 cells) | mouse | PCR_R.N2Ind12 |
RNA-ER | Mm ES (50 cells) | mouse | PCR_R.N2Ind13 |
Hap samples (1-12) prepared on lab note 6.6.11 [7]. Briefly, Twelve cell lysate aliquots (~1pg gDNA) were amplified with Nextera adapter mix (barcode 1-12) and mixed the 12 samples at 1:1 ratio.
RNA samples prepared on lab note [8]. Briefly, ES (~50cells) samples from Dr. Yi Zhang lab were directly lysated (named EL sample) or purified with RNA by Zymo kit (named ER sample), and then amplified with PCR_based protocol, which introduces poly(dT) to 5' end and poly(dA) to 3' end of the ds cDNA. Solexa Y adaptor was ligated to the ends and illumina barcoded primers (N2.ID12 and N2.ID13) were used to amplfied EL and ER samples, respectively.
Finally, Hap samples and RNA samples were mixed as 10:1:1 ratio for sequencing.
Manuals
Sequencing for Genewiz
Prepare for sequencing (Genewiz): 20ng DNA in 10ul total volume + 5ul 5uM primer
Zeroblunt cloning
- Vector map File:Zeroblunt vector map.pdf
- zeroblunt-topo Manual File:ZerobluntTOPO man.pdf
- zeroblunt manual File:Zeroblunt man.pdf
TA cloning
Vector map [9]
Manual File:Original TA cloning kit.pdf
Bio-Rad QPCR machine
- Program on desktop: "Opticon Moniter 3"
- Instrument: Chromo4:CD003042. "Instrument"-->"Scan for instruments" or "Quick load"(under "Master")-->choose "Chromo4:CD003042"
- "Prepare new run" or "open" a old file and "edit"
- "Plate setup"-->"edit"-->select "samples" and "SBG1" for SYBR green I
- "Protocol setup"-->"edit"-->add "plate read" before "repeat cycle"
- "Melting curve" optional, hold at 15C, volume 20ul
- "Run" and "Save", files are saved in "Opticon Users 3"/Rui
- "Status" and "Quantitation" are used for monitoring PCR process
- "Quantitation"-->"copy to clipboard"-->"Data graph" and edit under "Paint"
Ep Realplex QPCR machine
- "Mastercycler ep realplex" on desktop
- log in User name: "EPPENDORF", password: "e"
- "PCR program"-->direct change temp and time-->right click to insert and advance edit
- "Plate layout"-->"Filer 520nm"-->"SYBR"; "Sample volume", "Probe"-->"SYBR green", "Background"-->"Biorad strip tube-50ul" (Attention!); select sample area--> right click-->"unknown"-->ID
- "Save as" a "template" or an "assay"
- "Set up"-->"System Configuration"-->"Cycler"-->"Lid"-->select "Heat"-->Okey and close for preheat the lid
- "Monitoring" for PCR process
- "Analysis data" and save as assay