Rui:SingelCell: Difference between revisions

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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===
#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
* HRCA based: 1st try: 2.4.11
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name=Rui:LabNotes/SingleCell
format=%name/%year-%month-%day
date=2011/10/1
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Latest revision as of 00:51, 7 December 2011