Rui:SingelCell

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Revision as of 21:47, 9 January 2011 by >RuiLiu (→‎Single cell RNA isolation)
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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

Single cell RNA isolation

1. Single cell capture

-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
-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)
use fresh made working solution
Cell lysis buffer
use fresh made lysis buffer

2.1 Seed the cell into the 0.5 ml PCR tube with the lysis buffer [~5 ul as total]

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

PCR-based protocol

IVT-based protocol

Circulation-based protocol

2011

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