Rui:SingelCell RNAseq

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Revision as of 19:11, 14 November 2012 by >Jdeng (→‎PCR based protocol (Tang 2009))
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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

In vitro transcription based protocol

ABi RiboAmp HS kit

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

  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>