Rui:SingelCell

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Revision as of 19:25, 3 March 2011 by >RuiLiu (→‎ABi RiboAmp HS kit)
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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

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

step 1. 1st cDNA

Componet	V (ul)
RNA	        10
P1	        1
total	        11

65C, 90sec; chill on ice


42C, 1hr Componet V (ul) chill on ice Master mix 5 Enzyme mix 1 Enhancer 2 SSIII 1 Total 9

2. Nuclease mix 37C, 30min Componet V (ul) 95C, 5min Nuclease mix 2 chill on ice

3. 2nd cDNA 95C, 2min Componet V (ul) Chill on ice P2 1ul

25C, 10min Componet V (ul) 37C, 30min master mix 29 70C, 5min Enzyme mix 1 chill on ice Total 30

4. EtOH precipitate (-)80C, 1hr Componet V (ul) 14k rpm, 30min NaOAc 5.3 wash, airdry ice cold EtOH 132.5 resuspend 11ul 137.8

5. 1st round IVT 42C, 6hrs Componet V (ul) DNA 11 IVT buffer 2 IVT master mix 6 IVT Enzyme mix 2 Enhancer 2 Total 23

6. DNase mix 37C, 15min Componet V (ul) chill on ice DNase mix 1

7. aRNA purficiation Equilibrate column RT. 5min Componet V (ul) 16k g, 1min RB buffer 250

mix thoroughly Componet V (ul) 100 g, 2min aRNA 24 10k g, 1min RB buffer 120

Componet V (ul) 10k g, 1min RW 200

16k g, 2min Componet V (ul) 16k g, +1min RW 200

in 0.5mL tube Componet V (ul) RT, 1min RE 12 1k g, 1min 16k g, 1min

8. 1st cDNA 65C, 5min Componet V (ul) Chill on ice P2 1

25C, 10min Componet V (ul) 37C, 1hr Master mix 5 chill on ice Enzyme mix 1 Enhancer 2 SSIII 1 Total 9

9. 2nd cDNA 95C, 5min Componet V (ul) Chill on ice P3 1

37C, 30min Componet V (ul) 70C, 5min master mix 29 chill on ice Enzyme mix 1 Total 30

10. cDNA purification Equilibrate column RT. 5min Componet V (ul) 16k g, 1min DB buffer 250

mix thoroughly Componet V (ul) 100 g, 2min ds cDNA 52 10k g, 1min DB buffer 200

16k g, 2min Componet V (ul) 16k g, +1min DW 250

in 0.5mL tube Componet V (ul) RT, 1min DE 12 1k g, 1min 16k g, 1min

11. 2nd round IVT 42C, 6hrs Componet V (ul) DNA 11 IVT buffer 2 IVT master mix 6 IVT Enzyme mix 2 Enhancer 2 Total 23

12. DNase mix 37C, 15min Componet V (ul) chill on ice DNase mix 1

13. aRNA purficiation Equilibrate column RT. 5min Componet V (ul) 16k g, 1min RB buffer 250

mix thoroughly Componet V (ul) 100 g, 2min aRNA 24 10k g, 1min RB buffer 120

Componet V (ul) 10k g, 1min RW 200

16k g, 2min Componet V (ul) 16k g, +1min RW 200

in 0.5mL tube Componet V (ul) RT, 1min RE 12 1k g, 1min 16k g, 1min

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 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
  • HRCA based: 1st try: 2.4.11

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