Rui:SingelCell

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Revision as of 00:32, 11 January 2011 by >RuiLiu (→‎PCR 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

Protocols

PCR based protocol

Modified from Tang 2010, NProtocol, 5: 516

1. Single cell capture [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 [up to ~4.55 ul]

-Mild lysis buffer to ensure efficient lysis (thoroughly and completely lysed) w/o severely interfering with RT efficiency
-Positive control to establish the protocol at lab
-Negative control to rule out the contamination from the start
UP1 primer
sequence: ATATGGATCCGGCGCGCCGTCGACTTTTTTTTTTTTTTTTTTTTTTTT
stock solution: 100 uM
working solution: 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
Procedure
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 [up to ~5 ul]

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 [up to 6 ul]

5. 3' poly(A) tailing [up to 12 ul]

6. 2nd strand cDNA synthesis [up to 22 ul per tube, 4 tubes]

7. PCR amplification [up to 41 ul per tube, 4 tubes]

8. DNA purification [elute w/ 50 ul EB buffer]

IVT-based protocol

modified from RiboAmp HS PLUS Amplification Kit, ABi, Cat#

Circulation-based protocol

Nextera library preparation

2011

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