Rui:SingelCell RNAseq
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
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
- 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>