Rui:SingelCell: Difference between revisions
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===Single cell RNA isolation=== | ===Single cell RNA isolation=== | ||
1. Single cell capture | 1. Single cell capture | ||
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. | ||
2. Cell lysis | 2. Cell lysis | ||
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 | ||
===PCR-based protocol=== | ===PCR-based protocol=== |
Revision as of 21:08, 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
PCR-based protocol
IVT-based protocol
Circulation-based protocol
2011
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