Rui:SingelCell RNAseq: Difference between revisions

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>Jdeng
>Jdeng
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*[[Rui:Nextera based protocol|Nextera based protocol]]
*[[Rui:Nextera based protocol|Nextera based protocol]]
*[[Rui:totoRNAseq protocol|totoRNAseq protocol]]
*[[Rui:totoRNAseq protocol|totoRNAseq protocol]]
===In vitro transcription based protocol===
====ABi RiboAmp HS kit====
* Official protocol as in [[File:kit0525.pdf]]
'''step 1. 1st cDNA'''
'''Componet V (ul)'''
RNA         10
P1         1
total         11
65C, 90sec; chill on ice
'''Componet V (ul)'''
Master mix 5
Enzyme mix 1
Enhancer 2
SSIII         1
Total         9
42C, 1hr; chill on ice
'''Step2. Nuclease mix '''
'''Componet V (ul)'''
Nuclease mix 2
37C, 30min
95C, 5min
chill on ice
'''Step3. 2nd cDNA'''
'''Componet V (ul)'''
P2 1ul
95C, 2min
Chill on ice
'''Componet V (ul)'''
master mix 29
Enzyme mix 1
Total         30
25C, 10min
37C, 30min
70C, 5min
chill on ice
'''Step4. EtOH precipitate'''
'''Componet V (ul)'''
NaOAc         5.3
ice cold EtOH 132.5
Total 137.8
(-)80C, 1hr;
14k rpm, 30min;
wash, airdry;
resuspend 11ul
'''Step 5. 1st round IVT'''
'''Componet V (ul)'''
DNA         11
IVT buffer 2
IVT master mix 6
IVT Enzyme mix 2
Enhancer 2
Total         23
42C, 6hrs
'''Step6. DNase mix'''
'''Componet V (ul)'''
DNase mix 1
37C, 15min
chill on ice
'''Step7. aRNA purficiation'''
'''Componet V (ul)'''
RB buffer 250
Equilibrate column
RT. 5min
16k g, 1min
'''Componet V (ul)'''
aRNA         24
RB buffer 120
mix thoroughly
100 g, 2min
10k g, 1min
'''Componet V (ul)'''
RW         200
10k g, 1min
'''Componet V (ul)'''
RW         200
16k g, 2min
16k g, +1min
'''Componet V (ul)'''
RE         12
in 0.5mL tube
RT, 1min
1k g, 1min
16k g, 1min
'''Step8. 1st cDNA'''
'''Componet V (ul)'''
P2         1
65C, 5min
Chill on ice
'''Componet V (ul)'''
Master mix 5
Enzyme mix 1
Enhancer 2
SSIII         1
Total         9
25C, 10min
37C, 1hr
chill on ice
'''Step9. 2nd cDNA'''
'''Componet V (ul)'''
P3         1
95C, 5min
Chill on ice
'''Componet V (ul)'''
master mix 29
Enzyme mix 1
Total         30
37C, 30min
70C, 5min
chill on ice
'''Step10. cDNA purification'''
'''Componet V (ul)'''
DB buffer 250
Equilibrate column
RT. 5min
16k g, 1min
'''Componet V (ul)'''
ds cDNA 52
DB buffer 200
mix thoroughly
100 g, 2min
10k g, 1min
'''Componet V (ul)'''
DW         250
16k g, 2min
16k g, +1min
'''Componet V (ul)'''
DE         12
in 0.5mL tube
RT, 1min
1k g, 1min
16k g, 1min
'''Step11. 2nd round IVT'''
'''Componet V (ul)'''
DNA         11
IVT buffer 2
IVT master mix 6
IVT Enzyme mix 2
Enhancer 2
Total         23
42C, 6hrs
'''Step12. DNase mix'''
'''Componet V (ul)'''
DNase mix 1
37C, 15min
chill on ice
'''Step13. aRNA purification'''
same as Step7
====A-tailing-based P2 addition of ds cDNA IVT protocol====
*In vitro transcription requires a purified linear DNA template containing a promoter, ribonucleotide triphosphates, a buffer system that includes DTT and magnesium ions, and an appropriate phage RNA polymerase (T7, T3 or Sp6).
9.1 EtOH precipitation of ds cDNA reaction mixture to removal salt, primer and Taq (suspending DNA with 11ul Nf water)
9.2 IVT by using Arcturus RiboAmp HS PLUS Amplification Kit (ABi)
9.3 EtOH precipitation of aRNA before cDNA synthesis (suspending RNA with 8ul Nf water)
9.4 1st cDNA synthesis by using SS III first strand cDNA synthesis kit and P2 primer (Invitrogen)
9.5 2nd cDNA synthesis by DNA polymerase and P1 primer, modified from ds cDNA synthesis protocol (Invitrogen)
'''PCR reaction mix with mP1 primer'''
--------------------------------------------------------------
Componet Original [] Volume (ul) Final []
--------------------------------------------------------------
Ex Taq buffer 10*         15         1*
dNTP mix 10 mM          3         0.2 mM        
mP1 primer      0.5 uM        3.75         12.5 nM
Ex Taq HS    5 U/ul         1.5         0.05 U/ul
RNase H        2 U/ul        7.5            0.1 U/ul
N-F water         119.25
Total                 150        
--------------------------------------------------------------
9.6 EtOH precipitation of ds cDNA reaction mixture (suspending with 10ul N-f water)


===HRCA based protocol===
===HRCA based protocol===

Revision as of 19:11, 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>