Rui:SingelCell: Difference between revisions
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===IVT-based protocol=== | ===IVT-based protocol=== | ||
modified from RiboAmp HS PLUS Amplification Kit, ABi, Cat# | |||
===Circulation-based protocol=== | ===Circulation-based protocol=== | ||
Revision as of 21:54, 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
PCR based protocol
Modified from Tang 2010, NProtocol, 5: 516
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 [up to 5 ul]
-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 solution: 100 uM working solution: 0.5 uM (1 ul to 199 ul n-f water) use fresh made working solution
Cell lysis buffer use fresh made lysis buffer
2.1 Seed the cell into the 0.5 ml PCR tube with the lysis buffer
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
IVT-based protocol
modified from RiboAmp HS PLUS Amplification Kit, ABi, Cat#
Circulation-based protocol
2011
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