Rui:SingelCell

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Revision as of 00:35, 25 February 2011 by >RuiLiu (→‎HRCA based protocol)
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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
  • 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

Single cell capture

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.

Cell lysis

1. Cell lysis protocol [up to ~4.55 ul] modified from Tang 2010, NProtocol, 5: 516

-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'
Modified (72bp) T7 promoterpoly-BciVI-(dT): 5' AAACGACGGCCAGTGAATTGTAATACGACTCACTATAGGCGC-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

1st strand cDNA synthesis

3.1. 1st strand cDNA synthesis [up to ~5 ul] followed Tang 2010, NProtocol, 5: 516

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
3.1.1 Prepare RT mix and add 0.45 to each tube
3.1.2 Incubate at 50C for 30min
3.1.3 Inactivate the reverse transcriptase at 70C for 15min
3.1.4 Centrifuge tubes for 30s at 7,500g at 4C
3.1.5 Put on ice immediately for 1min 

3.2. Starting directly with total RNA [up to 5ul]

--------------------------------------------------------------
Componet	 Original []	Volume (ul)	Final []
-------------------------------------------------------------- 
total RNA                      0.5
PCR buffer II	 10*	        0.5	        1*
MgCl2	         25 mM	        0.27	        1.35 mM
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
mP1 primer	 0.5 uM	        0.125	        12.5 nM
dNTP mix	 2.5 mM each	0.09	        0.045 mM each
SuperScript III 200 U/ul	0.33            12.2 U/ul
T4 gene 32	 1-10 U/ul      0.07	        0.07 U/ul
N-F water		        2.8	
Total		                5ul	        
--------------------------------------------------------------

3.2.1 Mix total RNA with Primer and dNTP (water) 
3.2.2 Denature at 65C for 5min and put on ice immediately for 1min
3.2.3 Prepare the rest of the master mix and add to each tube
3.2.4 Incubate at 50C for 30min
3.2.5 Inactivate the reverse transcriptase at 80C for 5min
3.2.6 Centrifuge tubes for 30s at 7,500g at 4C
3.2.7 Put on ice immediately for 1min

Free primer removal

4.1 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
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

3' poly(A) tailing

5.1 A tailing followed Tang 2010 [up to 12 ul]

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

2nd strand cDNA synthesis

6.1 2nd strand cDNA synthesis modified from Tang 2010 [up to 22 ul per tube, 4 tubes]

P2 primer
Original (48bp): 5' ATATCTCGAGGGCGCGCCGGATCCTTTTTTTTTTTTTTTTTTTTTTTT
Modified (54bp) Anchor-BciVI-AdT: 5' ATATCTCGAGGGCGCGCCGGATCC-GTATCC-TTTTTATTTTTTTTTTTTTTTTTT
stock solution: 100 uM

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	        
--------------------------------------------------------------
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

PCR Amplification

7.1 PCR-based exponential amplification followed Tang 2010 [up to 41 ul per tube, 4 tubes]

PCR reaction mix with mP1 primer
--------------------------------------------------------------
Componet	 Original []	Volume (ul)	Final []
-------------------------------------------------------------- 
Ex Taq buffer	 10*	        7.6	        1*
dNTP mix	 2.5 mM         7.6	        0.25 mM	        
UP1 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	        
--------------------------------------------------------------
7.1.1 Prepare PCR mix and add 19 ul to each tube
7.1.2 PCR cycle: 95C 30s; 67C 1min; 72C 6min (+6s per cycle); 20 cycle; 4C hold
7.1.3 Centrifuge tube for 30s at 7,500g at 4C
7.1.4 Store at -80 up to 6 months

7.2 PCR-based exponential amplification by KAPA SYBR® FAST qPCR Kits

QC and DNA purification [10 EB buffer]

8.1 Combine the 4 tubes for each sample
8.2 Take 10 ul mix for QC
    1~2ul for cloning and Sanger sequencing
    1~2ul of 10-fold dilute for a 20ul SYBR Green qPCR (GAPDH, HPRT, ACTB etc.) for RNA quantity and DNA contamination
    Rest: gel check for size and quantity
8.3 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

In vitro transcription

  • 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)

BciVI digestion

set up a digestion reaction around 15~20ul

Concatemerization

set up ligation reaction w/o purification around ~50ul

Circulation

  1. Cell lysis
  2. 1st cDNA synthesis
  3. Exo I treatment
  4. RNase A/H/T1 treatment
  5. Circulation
  6. HRCA with Bst large fragment

Nextera library preparation

Outline

PCR based protocol

  • 1st try: 1.24.11
  • Trouble shooting: 1.26.11
  • 2nd try: 2.7.11
  • dNTP modification: 2.14.11

IVT based protocol

  • 1st try: 2.8.11

HRCA based protocol

  • 1st try: 2.4.11

2011

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