Rui:SingelCell: Difference between revisions
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**Maintain the integrity of living single cells before lysis (dissociation - transfer - wash - transfer) | **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. | **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=== | ===PCR-based protocol=== |
Revision as of 21:05, 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
- 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
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