Rui:SingelCell RNAseq: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
>Jdeng
>Jdeng
Line 21: Line 21:
*[[Rui:IVT note|IVT note]]
*[[Rui:IVT note|IVT note]]
*[[Rui:HRCA note|HRCA note]]
*[[Rui:HRCA note|HRCA note]]
===PCR note===
* Full text in PCR lab note.xls file
{| {{table}} border=1
| align="center" style="background:#f0f0f0;"|'''PCR_based'''
| align="center" style="background:#f0f0f0;"|'''Description'''
| align="center" style="background:#f0f0f0;"|'''Key elements'''
| align="center" style="background:#f0f0f0;"|'''start'''
| align="center" style="background:#f0f0f0;"|'''result'''
| align="center" style="background:#f0f0f0;"|'''comments'''
|-
| 1st try||Original protocol||P1||1.24.11||1.25.11||undistinguishable
|-
| ||Trouble shooting||QPCR step||1.26.11||1.26.11||
|-
| ||||Clone||1.26.11||2.2.11||
|-
| ||||ExoI adjustment||1.27.11||2.4.11||
|-
| 2nd try||ExoI; P8||usb ExoI;P8;Kapa||2.7.11||2.8.11||Amp (+); BK (-)
|-
| ||||usb ExoI;P1;Kapa||2.9.11||||
|-
| ||after PCR||Purif. Lig. Clone||2.10.11||2.17/24.11||
|-
| 3rd try||Adjust [dNTP]||45uM; 0.45uM; 4.5nM||2.14.11||2.15.11||Failed
|-
| ||||repeat||2.17.11||2.18.11||
|-
| ||[UP1] & [dNTP]||12.5/3.5nM; 45/10uM||3.10.11||3.11.11||Reduced size range
|-
| ||||size select and clone||3.14.11||3.18.11||
|-
| ||UP1/P8 & [dNTP] ||12.5/3.5nM; 45/10/2.5uM||3.15.11||3.16.11||Reduced size range
|-
| ||||repeat||3.17.11||||Failed
|-
| 4th try||dATP||3mM vs 1mM||3.21.11||3.22.11||3mM better
|-
| ||||size and clone||3.25.11||3.28.11||
|-
| ||||dNTP, P8 vs P9||3.29.11||||
|-
| ||||size and clone||3.30.11||4.1.11||
|-
| 5th try||NTC in every step||all NTC for each step||3.29.11||3.29.11||RT primer + A-tail
|-
|
|-
| 6th try||ddATP||1:20 ddATP:dATP||3.31.11||||Distinguished
|-
| ||||TBU||4.4.11||||
|-
| ||||repeat||4.5.11||4.6.11||
|-
| ||||repeat 2nd half||4.11.11||4.12.11||
|-
| ||||size and clone||4.12.11||4.16.11||
|-
| ||2nd PCR||2nd round PCR||4.13.11||||
|-
| ||||purify and clone||4.18.11||4.21.11||
|-
| 7th try||poly(dT) in RT||test for 6 PCR primers||4.28.11||4.28.11||Great!
|-
| ||confirm with RNAs||poly(dT); P8||4.29.11||4.29.11||Bravo!
|-
| ||||Digestion; ligation; Phusion||5.2.11||5.4.11||No good - adjust!
|-
| 8th try||Lymphocytes||10X more P8||5.9.11||5.9.11||
|-
| ||||repeat||5.10.11||5.10.11||Ctrl failed
|-
| ||||new ctrl, gDNA, cell lysates||5.11.11||5.12.11||gDNA contamination
|-
| ||||Cloning||5.13.11||5.16.11||Summary on 5.17
|-
| ||||Digestion; ligation; Phusion||5.17.11||5.18.11||5.18.11
|}
{| {{table}} border=1
| align="center" style="background:#f0f0f0;"|'''50 cells RNAseq'''
| align="center" style="background:#f0f0f0;"|'''RNA or lysate'''
| align="center" style="background:#f0f0f0;"|'''Sperm vs ES cells'''
| align="center" style="background:#f0f0f0;"|'''5.19.11'''
| align="center" style="background:#f0f0f0;"|'''5.20.11'''
| align="center" style="background:#f0f0f0;"|''''''
|-
| ||||process, clone||5.21.11||5.24.11||5.25.11 seq result
|-
| ||||Nextera library||5.23.11||5.23.11||spread out (clone?)
|-
| ||||Solexa library||5.23.11||5.23.11||amp. Not good
|-
| ||||||5.26.11||5.26.11||again. Not good
|-
| ||||||6.1.11||6.2.11||different buffs
|-
| ||||||6.2.11||6.3.11||another 2ug repeat
|-
| ||||||6.4.11||||Kapa for NGS
|}


===IVT note===
===IVT note===

Revision as of 19:14, 14 November 2012

Background

  • Goal: the smallest functional unit; the exact quantity; full-length RNAs; sense vs anti-sense; all classes; at single-base resolution
  • Review papers:
  • Key papers:
  1. Single cell and cell lysis
  2. 1st cDNA synthesis and free primer removal
  3. 2nd cDNA synthesis: A-tailing vs random
  4. Amplification: PCR or IVT
  5. QC and clone

Protocols

QuickNotes

IVT note

IVT_based description start result
1st try 3 sets 2.8.11 2.9.11
Following purification? 2.10.11
primer check 2.15.11
clone 2.17.11 2.24.11
clone troubleshoot 2.28.11
2nd round IVT 3.2.11 3.3.11
trouble shooting 3.3.11 3.4.11
Atailed_ds cDNA IVT 3.2.11 3.3.11
2nd try repeat kit 3.8.11 3.9.11
3.10.11 3.11.11
Atailed_ds cDNA IVT 4.5.11 4.6.11


HRCA note

HRCA description start result
1st try P1 2.3.11 2.4.11

2012

<calendar> name=Rui:LabNotes/SingleCell format=%name/%year-%month-%day date=2012/10/1 view=oneyear </calendar>


2011

<calendar> name=Rui:LabNotes/SingleCell format=%name/%year-%month-%day date=2011/10/1 view=oneyear </calendar>