Rui:SingelCell: Difference between revisions
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'''Cell lysis buffer''' | '''Cell lysis buffer''' | ||
2.1 Seed the cell into the 0.5 ml PCR tube with the lysis buffer | 2.1 Seed the cell into the 0.5 ml PCR tube with the lysis buffer [~5 ul as total] | ||
2.2 Centrifuge | 2.2 Centrifuge for 30s at 7,500g at 4C | ||
2.3 Put on ice immediately | 2.3 Put on ice immediately | ||
2.4 Incubate at 70C for 90s | |||
2.5 Put on ice immediately | |||
2.6 Centrifuge for 30s at 7,500g at 4C | |||
2.7 Put on ice immediately for 1min | |||
===PCR-based protocol=== | ===PCR-based protocol=== |
Revision as of 21:44, 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
2.1 Seed the cell into the 0.5 ml PCR tube with the lysis buffer [~5 ul as total]
2.2 Centrifuge for 30s at 7,500g at 4C
2.3 Put on ice immediately
2.4 Incubate at 70C for 90s
2.5 Put on ice immediately
2.6 Centrifuge for 30s at 7,500g at 4C
2.7 Put on ice immediately for 1min
PCR-based protocol
IVT-based protocol
Circulation-based protocol
2011
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