Rui:SingelCell RNAseq: Difference between revisions
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>Jdeng |
>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:
- Single cell and cell lysis
- 1st cDNA synthesis and free primer removal
- 2nd cDNA synthesis: A-tailing vs random
- Amplification: PCR or IVT
- QC and clone
Protocols
- PCR based protocol
- IVT based protocol
- HRCA based protocol
- Nextera based protocol
- totoRNAseq protocol
HRCA based protocol
- Cell lysis
- PNK P1
- 1st cDNA synthesis
- Exo I treatment
- RNase A/H/T1 treatment
- Circulation
- 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>