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==
===PCR based protocol===
Modified from Tang 2010, ''NProtocol'', 5: 516
1. Single cell capture [up to ~0.1 ul]
-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 ~4.55 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)
directly add into lysis buffer?
'''Cell lysis buffer'''
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Componet'''
| align="center" style="background:#f0f0f0;"|'''Original concentration'''
| align="center" style="background:#f0f0f0;"|'''Volume (ul)'''
| align="center" style="background:#f0f0f0;"|'''Final concentration'''
|-
| PCR buffer II||10*||0.45||0.9*
|-
| MgCl2||25 mM||0.27||1.35 mM
|-
| NP40||10%||0.225||0.45%
|-
| DTT||0.1 M||0.225||4.5 mM
|-
| SUPERase-In ||20 U/ul||0.045||0.18 U/ul
|-
| RNase inhibitor||40 U/ul||0.045||0.36 U/ul
|-
| UP1 primer||0.5 uM||0.125||12.5 nM
|-
| dNTP mix||2.5 mM each||0.09||0.045 mM each
|-
| N-F water||||2.975||
|-
| Total||||4.45||in 5ul
|}
use fresh made lysis buffer
'''Procedure'''
2.1 Seed the cell into the 0.5 ml PCR tube with 4.45 ul 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 1m
3. 1st strand cDNA synthesis [up to ~5 ul]
4. Free primer removal [up to 6 ul]
5. 3' poly(A) tailing [up to 12 ul]
6. 2nd strand cDNA synthesis [up to 22 ul per tube, 4 tubes]
7. PCR amplification [up to 41 ul per tube, 4 tubes]
8. DNA purification [elute w/ 50 ul EB buffer]
===IVT-based protocol===
modified from RiboAmp HS PLUS Amplification Kit, ABi, Cat#
===Circulation-based protocol===
===Nextera library preparation===
==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