Rui:SingelCell

From ZhangLabWiki
Revision as of 21:05, 9 January 2011 by >RuiLiu (→‎Single cell RNA isolation)
Jump to navigation Jump to search

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

  • 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.
  • 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

PCR-based protocol

IVT-based protocol

Circulation-based protocol

2011

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