Rui:SingelCell: Difference between revisions

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==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 [PBS-BSA carryover - 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
===PCR-based protocol===
===IVT-based protocol===
===Circulation-based protocol===
==2011==
<calendar>
name=Rui:LabNotes/SingleCell
format=%name/%year-%month-%day
date=2011/10/1
view=oneyear
</calendar>

Latest revision as of 00:51, 7 December 2011