Rui:SingelCell RNAseq: Difference between revisions

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>Jdeng
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#Circulation
#Circulation
#HRCA with Bst large fragment
#HRCA with Bst large fragment


===Nextera kit===
===Nextera kit===

Revision as of 19:12, 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

HRCA based protocol

  1. Cell lysis
  2. PNK P1
  3. 1st cDNA synthesis
  4. Exo I treatment
  5. RNase A/H/T1 treatment
  6. Circulation
  7. HRCA with Bst large fragment

Nextera kit

manual File:Nextera samp prep.pdf

QuickNotes

PCR note

  • Full text in PCR lab note.xls file
PCR_based Description Key elements start result 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
50 cells RNAseq RNA or lysate Sperm vs ES cells 5.19.11 5.20.11 '
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_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>