Rui:SingelCell: Difference between revisions

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2.1 Seed the cell into the 0.5 ml PCR tube with the lysis buffer
2.1 Seed the cell into the 0.5 ml PCR tube with the lysis buffer
2.2 Centrifuge the tube for 30s at 7,500g at 4C
2.2 Centrifuge the tube for 30s at 7,500g at 4C
2.3 Put on ice immediately
2.3 Put on ice immediately



Revision as of 21:41, 9 January 2011

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 concentration: 100 uM
working concentration: 0.5 uM (1 ul to 199 ul n-f water)
Cell lysis buffer

2.1 Seed the cell into the 0.5 ml PCR tube with the lysis buffer

2.2 Centrifuge the tube for 30s at 7,500g at 4C

2.3 Put on ice immediately

PCR-based protocol

IVT-based protocol

Circulation-based protocol

2011

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