Rui:LabNotes/SingleCell/2013-7-31: Difference between revisions
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>RuiLiu m (→Procedures) |
>RuiLiu m (→Results) |
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===Results=== | ===Results=== | ||
[[File:8.1.13_IVT-TSO.jpg]] | |||
===Libraries=== | ===Libraries=== |
Revision as of 01:38, 2 August 2013
mNPC plate with the latest version of hiMg-TSO and hiMg-IVT protocols
- The latest version of hiMg-TSO and hiMg-IVT protocols confirmed on 7/29/13
- I didn't use Betaine because it hasn't come in yet
- mNPC plate was sorted on 7.10.13 in hiMg buffer [1]
Design
Procedures
- 2ul Lysis: the long procedure originally used for fluidigm
- 3ul PNK: 1mM ATP, RNase-In (1:2d), PNK (1:2d)
- 4ul PAP: 1mM ATP, PAP (1:10d)
- 10ul RT: 3ul 0.2uM T20.id01-24 or RL.T7.id01-24
- beads pools into 4 tubes, 3 column each (240ul beads + 240ul rec.)
- TSO with TSO.r04 or TSO.r05 (LNA)
- 2nd strand in 20ul
- T7 addition for T20.id01-24 for 2 step IVT
- beads purification
- IVT in 20ul for 12 hrs
- Directly take 2ul for a quick run (RT-PCR), purify the rest with Zymo kit
- RT with N6 or N9
- QPCR for 6-9 cycles
- Redo RT-PCR with adjusted aRNA input
- beads purification or size selection (if primer-dimer is an issue)