Rui:SingelCell: Difference between revisions

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  *Mild lysis buffer to ensure efficient lysis (thoroughly and completely lysed) w/o severely interfering with RT efficiency
  *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
  *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


===PCR-based protocol===
===PCR-based protocol===

Revision as of 21:14, 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

PCR-based protocol

IVT-based protocol

Circulation-based protocol

2011

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