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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