Rui:SingelCell
Background
- Goal: the smallest functional unit; the exact quantity; full-length RNAs; sense vs anti-sense; all classes; at single-base resolution
- Step 1: single cell; exact quantity; the 3' end; mRNA; RNAseq
PCR based protocol
- 1. Single cell
- 2. Cell lysis
- 3. 1st strand cDNA synthesis
- 4. Free primer removal
- 5. 3' poly(dA) tailing
- 6. 2nd strand cDNA synthesis
- 7. PCR amplification
- 8. Library QC
- 9. EtOH precipitation
- 10. BciVI digestion
- 11. Beads purification to remove ends
- 12. Ligation
- 13. EtOH precipitation
- 14. Nextera lib prep
IVT based protocol
- 1. Single cell
- 2. Cell lysis
- 3. 1st strand cDNA synthesis with P1
- 4. Free primer removal
- 5. 3' poly(dA) tailing
- 6. 2nd strand cDNA synthesis with P2
- 7. EtOH precipitation
- 8. IVT
- 9. EtOH precipitation
- 10. 1st strand cDNA synthesis with P2
- 11. 2nd strand cDNA synthesis with P1
- 12. Library QC
- 13. EtOH precipitation
- 14. BciVI digestion
- 15. Beads purification
- 16. Ligation
- 17. EtOH precipitation
- 18. Nextera lib prep
Circulation
- 1. Single cell
- 2. Cell lysis
- 3. PNK P1
- 4. 1st strand cDNA synthesis
- 5. Exonuclease I treatment
- 6. RNase A/H/T1 treatment
- 7. Circulation by CircLigase
- 8. RCA with P3 and P4
- 9. Library QC
- 10. EtOH precipitation
- 11. BciVI digestion
- 12. Beads purification
- 13. Ligation
- 14. EtOH precipitation
- 15. Nextera lib prep
Protocols
PCR based protocol (Tang 2009)
1. Single cell capture by mouth pipetting [up to ~0.1 ul]
- Maintain the integrity of living single cells before lysis (dissociation - transfer - wash - transfer)
- PBS alone will cause tight attachment of cells to dish/micropipette, leading to rupture/loss of cells.
2. Cell lysis
- Mild lysis buffer to ensure efficient lysis (thoroughly and completely lysed) w/o severely interfering with RT efficiency
- Positive control to establish the protocol (total RNA, 10pg, 50pg, 100pg, 500pg)
- Negative control to rule out the contamination from the start
P1 primer Original (48bp): 5' ATATGGATCCGGCGCGCCGTCGACTTTTTTTTTTTTTTTTTTTTTTTT 3' Rui-P1 (72nt) extra-T7promoter-BciVI-poly(dT): 5' AAACGACGGCCAGTGAATTGTAATACGACTCACTATAGGCGC-GTATCC-TTTTTTTTTTTTTTTTTTTTTTTT 3' Rui-P8 (51nt) T7promoter-BciVI-poly(dT): 5' TAATACGACTCACTATAGGG-GTATCC-TTTTTTTTTTTTTTTTTTTTTTTT 3' stock solution: 100 uM working solution for RT: 0.5 uM (1 ul to 199 ul n-f water)
Cell lysis buffer -------------------------------------------------------------- Componet Original [] Volume (ul) Final [] -------------------------------------------------------------- PCR buffer II 10* 0.45 0.9* MgCl2 25 mM 0.27 1.35 mM NP40 10% 0.225 0.45% DTT 0.1 M 0.225 4.5 mM SUPERase-In 20 U/ul 0.045 0.18 U/ul RNase inhibitor 40 U/ul 0.045 0.36 U/ul UP1 primer 0.5 uM 0.125 12.5 nM dNTP mix 2.5 mM each 0.09 0.045 mM each N-F water 2.975 Total 4.45 in 5ul -------------------------------------------------------------- use fresh made lysis buffer
2.1 Seed the cell into the 0.5 ml PCR tube with 4.45 ul lysis buffer 2.2 Centrifuge for 30s at 7,500g at 4C 2.3 Put on ice immediately 2.4 Incubate at 70C for 90s 2.5 Put on ice immediately 2.6 Centrifuge for 30s at 7,500g at 4C 2.7 Put on ice immediately for 1min
3. 1st strand cDNA synthesis
RT reaction mix ---------------------------------------------------------------------------------- Componet Original [] Volume (ul) Final [] ---------------------------------------------------------------------------------- SuperScript III reverse transcriptase 200 U/ul 0.33 12.2 U/ul RNase inhibitor 40 U/ul 0.05 0.4 U/ul T4 gene 32 protein 1-10 U/ul 0.07 0.07 U/ul Total 0.45 ---------------------------------------------------------------------------------- Prepare and maintain on ice before use
3.1 Prepare RT mix and add 0.45 to each tube 3.2 Incubate at 50C for 30min 3.3 Inactivate the reverse transcriptase at 70C for 15min 3.4 Centrifuge tubes for 30s at 7,500g at 4C 3.5 Put on ice immediately for 1min
4. Free primer removal
- Free primer removal followed Tang 2010 [up to 6 ul]
Exonulease I mix ---------------------------------------------------------------------------------- Componet Original [] Volume (ul) Final [] ---------------------------------------------------------------------------------- Exonuclease I buffer 10* 0.1 1* N-f Water 0.8 Exonuclease 5 U/ul 0.1 0.5 U/ul Total 1 ---------------------------------------------------------------------------------- Prepare and maintain on ice before use
- Free primer removal based on experimental evaluation on ExoI efficiency
Exonulease I mix ---------------------------------------------------------------------------------- Componet Original [] Volume (ul) Final [] ---------------------------------------------------------------------------------- N-f Water 0-fill up to 1 Exonuclease I (USB) 10 U/ul 0.2-0.5 2-5 U ----------------------------------------------------------------------------------
- Free primer removal based on ABi RiboAmp HS kit
Exonulease mix ---------------------------------------------------------------------------------- Componet Original [] Volume (ul) Final [] ---------------------------------------------------------------------------------- Exonuclease VIII Exonuclease I (USB) 10 U/ul 0.2-0.5 2-5 U ----------------------------------------------------------------------------------
4.1 Prepare Exonulease I mix and add 1 ul to each tube 4.2 Incubate at 37C for 30min 4.3 Inactivate the reaction at 80C for 25min 4.4 Centrifuge the tube for 30sec at 7,500g at 4C 4.5 Put on ice immediately for 1min
5. 3' poly(A) tailing
TdT reaction mix -------------------------------------------------------------- Componet Original [] Volume (ul) Final [] -------------------------------------------------------------- PCR buffer II 10* 0.6 1* MgCl2 25 mM 0.36 1.5 mM dATP 100 mM 0.18 3 mM RNase H 2 U/ul 0.3 0.1 U/ul TdT 15 U/ul 0.3 0.75 U/ul N-F water 4.26 Total 6 --------------------------------------------------------------
5.1 Prepare TdT mix and add 6ul to each tube 5.2 Incubate at 37C for 15min 5.3 Inactivate the TdT at 70C for 10min 5.4 Centrifuge for 30s at 7,500g at 4C 5.5 Put on ice immediately for 1 min
6. 2nd strand cDNA synthesis
P2 primer Original (48bp): 5' ATATCTCGAGGGCGCGCCGGATCCTTTTTTTTTTTTTTTTTTTTTTTT Modified (54bp) Anchor-BciVI-AdT: 5' ATATCTCGAGGGCGCGCCGGATCC-GTATCC-TTTTTATTTTTTTTTTTTTTTTTT stock solution: 100 uM
- PCR-based exponential amplification followed Tang 2010
PCR reaction mix with mP2 primer -------------------------------------------------------------- Componet Original [] Volume (ul) Final [] -------------------------------------------------------------- Ex Taq buffer 10* 7.6 1* dNTP mix 2.5 mM 7.6 0.25 mM mP2 primer 100 uM 0.76 1 uM Ex Taq HS 5 U/ul 0.76 0.05 U/ul N-F water 59.28 Total 76 --------------------------------------------------------------
- PCR-based exponential amplification by KAPA SYBR® FAST qPCR Kits
-------------------------------------------------------------- Componet Original [] Volume (ul) Final [] -------------------------------------------------------------- P1/8 primer 100 uM 0.76 1 uM Kapa-mix 2* 44 1* N-F water 31.24 Total 76 --------------------------------------------------------------
6.1 Divide TdT reaction mix into 4 tubes with 3 ul per tube 6.2 Prepare PCR mix and add 19 ul to each tube 6.3 PCR cycle: 95C 3min; 50C 2min; 72C 10min; 1 cycle 6.4 Put on ice for 1min 6.5 Centrifuge tube for 30s at 7,500g at 4C 6.6 Put on ice immediately for 1min
7. PCR Amplification
- PCR-based exponential amplification followed Tang 2010 [up to 41 ul per tube, 4 tubes]
PCR reaction mix with P1 or P8 primer -------------------------------------------------------------- Componet Original [] Volume (ul) Final [] -------------------------------------------------------------- Ex Taq buffer 10* 7.6 1* dNTP mix 2.5 mM 7.6 0.25 mM P1/8 primer 100 uM 0.76 1 uM Ex Taq HS 5 U/ul 0.76 0.05 U/ul N-F water 59.28 Total 76 --------------------------------------------------------------
- PCR-based exponential amplification by KAPA SYBR® FAST qPCR Kits
-------------------------------------------------------------- Componet Original [] Volume (ul) Final [] -------------------------------------------------------------- P1/8 primer 100 uM 0.76 1 uM Kapa-mix 2* 44 1* N-F water 31.24 Total 76 --------------------------------------------------------------
7.1 Prepare PCR mix and add 19 ul to each tube 7.2 PCR cycle: 95C 30s; 67C 1min; 72C 6min (+6s per cycle); 20 cycle; 4C hold 7.3 Centrifuge tube for 30s at 7,500g at 4C 7.4 Store at -80 up to 6 months
8. DNA precipitation, digestion and purification
8.1 Combine the 4 tubes for each sample 8.2 Purify the remaining PCR product by EtOH precipitation and suspend w/ 10 ul EB buffer Add 1/10 V 3M NaOAc, 0.05-1μg/μl glycogen, and 2.5 V EtOH, additional PCR primers to each tube Incubate at -80 for 1hr Centrifuge at 10,000g for 30min Wash w/ ice-cold 70% EtOH 8.3 BciVI digestion set up a digestion reaction around 15~20ul 8.4 USB SAP-IT treatment to remove the primers and de-phosphate ds DNA for half of hour 8.5 Beads purification (0.8*)
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 library preparation
2011
Headsup
- PCR based:
1st try: 1.24.11
Trouble shooting: 1.26.11
2nd try: 2.7.11
dNTP modification: 2.14.11
- IVT based:
1st try: 2.8.11
2nd round: 3.3.11
- HRCA based:
1st try: 2.4.11
- Cloning and trouble shooting
PCR products: 2.2.11; 2.11.11; 2.24.11
IVT products: 2.16.11; 2.17.11; 2.24.11; 2.28.11
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