Rui:SingelCell RNAseq: Difference between revisions

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==SCAP project==
* SCAP website shared among UCSD/ILMN/Scripps, the main link [http://genome-tech.ucsd.edu/SCAP/index.php/Main_Page] pwd:"24"
* Sample's link: Data center - under SCAP website, SingleCell, SingleCell!@34
* Libraries [http://genome-tech.ucsd.edu/LabNotes/index.php/Rui:RNAseq-libraries]
* Data: genome-miner.ucsd.edu:/media/Ext4T/KZ_Ext4T/RNAseq/
* RNAseq pipeline http://genome-tech.ucsd.edu/LabNotes/index.php/RNAseq_pipeline [http://genome-tech.ucsd.edu/LabNotes/index.php/RNAseq_pipeline]
==Background==
==Background==
* Goal: the smallest functional unit; the exact quantity; full-length RNAs; sense vs anti-sense; all classes; at single-base resolution
* Goal: the smallest functional unit; the exact quantity; full-length RNAs; sense vs anti-sense; all classes; at single-base resolution
Line 11: Line 18:


==Protocols==
==Protocols==
===PCR based protocol (Tang 2009)===
*[[Rui:PCR based protocol|PCR based protocol]]
1. Single cell capture by mouth pipetting [up to ~0.1 ul]
*[[Rui:IVT based protocol|IVT based protocol]]
* Maintain the integrity of living single cells before lysis (dissociation - transfer - wash - transfer)
*[[Rui:HRCA based protocol|HRCA based protocol]]
* PBS alone will cause tight attachment of cells to dish/micropipette, leading to rupture/loss of cells.
*[[Rui:Nextera based protocol|Nextera based protocol]]
 
*[[Rui:toto-TSO_RNAseq protocol|toto-TSO_RNAseq protocol]]
2. Cell lysis
*[[Rui:toto-IVT_RNAseq protocol|toto-IVT_RNAseq protocol]]
* 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 kit===
manual [[File:Nextera_samp_prep.pdf]]


==QuickNotes==
==QuickNotes==
*[[Rui:PCR note|PCR note]]
*[[Rui:IVT note|IVT note]]
*[[Rui:HRCA note|HRCA note]]
*[[Rui:10.10.13 discussion|10.10.13 discussion]]


===PCR note===
* Full text in PCR lab note.xls file
{| {{table}} border=1
| align="center" style="background:#f0f0f0;"|'''PCR_based'''
| align="center" style="background:#f0f0f0;"|'''Description'''
| align="center" style="background:#f0f0f0;"|'''Key elements'''
| align="center" style="background:#f0f0f0;"|'''start'''
| align="center" style="background:#f0f0f0;"|'''result'''
| align="center" style="background:#f0f0f0;"|'''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
|}
{| {{table}} border=1
| align="center" style="background:#f0f0f0;"|'''50 cells RNAseq'''
| align="center" style="background:#f0f0f0;"|'''RNA or lysate'''
| align="center" style="background:#f0f0f0;"|'''Sperm vs ES cells'''
| align="center" style="background:#f0f0f0;"|'''5.19.11'''
| align="center" style="background:#f0f0f0;"|'''5.20.11'''
| align="center" style="background:#f0f0f0;"|''''''
|-
| ||||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===


{| {{table}} border=1
==2014==
| align="center" style="background:#f0f0f0;"|'''IVT_based'''
* Reference RNA data analysis:  
| align="center" style="background:#f0f0f0;"|'''description'''
* The first 1000 single-nucleus data workflow: 7.3; 7.4; 7.14; 8.7
| align="center" style="background:#f0f0f0;"|'''start'''
* The bulk sample analysis: 7.9.14; 10.10.14
| align="center" style="background:#f0f0f0;"|'''result'''
* The pipeline:
|-
| 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
|}


<calendar>
name=Rui:LabNotes/SingleCell
format=%name/%year-%month-%day
date=2014/10/1
view=oneyear
</calendar>


==2013==
<calendar>
name=Rui:LabNotes/SingleCell
format=%name/%year-%month-%day
date=2013/10/1
view=oneyear
</calendar>


===HRCA note===
==2012==
{| {{table}} border=1
<calendar>
| align="center" style="background:#f0f0f0;"|'''HRCA'''
name=Rui:LabNotes/SingleCell
| align="center" style="background:#f0f0f0;"|'''description'''
format=%name/%year-%month-%day
| align="center" style="background:#f0f0f0;"|'''start'''
date=2012/10/1
| align="center" style="background:#f0f0f0;"|'''result'''
view=oneyear
|-
</calendar>
| 1st try||P1||2.3.11||2.4.11
|}


==2011==
==2011==

Latest revision as of 06:14, 13 October 2014

SCAP project[edit]

Background[edit]

  • 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:
  1. Single cell and cell lysis
  2. 1st cDNA synthesis and free primer removal
  3. 2nd cDNA synthesis: A-tailing vs random
  4. Amplification: PCR or IVT
  5. QC and clone

Protocols[edit]

QuickNotes[edit]


2014[edit]

  • Reference RNA data analysis:
  • The first 1000 single-nucleus data workflow: 7.3; 7.4; 7.14; 8.7
  • The bulk sample analysis: 7.9.14; 10.10.14
  • The pipeline:

<calendar> name=Rui:LabNotes/SingleCell format=%name/%year-%month-%day date=2014/10/1 view=oneyear </calendar>

2013[edit]

<calendar> name=Rui:LabNotes/SingleCell format=%name/%year-%month-%day date=2013/10/1 view=oneyear </calendar>

2012[edit]

<calendar> name=Rui:LabNotes/SingleCell format=%name/%year-%month-%day date=2012/10/1 view=oneyear </calendar>

2011[edit]

<calendar> name=Rui:LabNotes/SingleCell format=%name/%year-%month-%day date=2011/10/1 view=oneyear </calendar>