AlanFung:LabNotes/CTCF/2011-6-28: Difference between revisions
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==Objective== | ==Objective== | ||
*Work on sperm/ESCs control cell samples plus GM12878 gDNA | *Work on sperm/ESCs control cell samples plus GM12878 gDNA | ||
#[[Sample List]] | |||
#A1-100 sperm cells | #A1-100 sperm cells | ||
#A2-100 sperm cells (repeat) | #A2-100 sperm cells (repeat) |
Revision as of 00:14, 28 June 2011
MDA Bisulfite Converted Sequencing Library Consturction
Objective
- Work on sperm/ESCs control cell samples plus GM12878 gDNA
- Sample List
- A1-100 sperm cells
- A2-100 sperm cells (repeat)
- B1-100 ESCs
- B2-100 ESCs (repeat)
- C-600pg GM12878 gDNA
- D-1.2ng GM12878 gDNA
- E-NTC
Overview
- Proteinase K Digestion
- Bisulfite conversion
- MDA
- EtOH precipitation
- Nextera Library Construction (Low input protocol)
- Page Analysis
- Sanger Sequencing
Protocol
Proteinase K Digestion
- Sperms: ~50cell/tube; 15 tubes
- ES cells: ~50cell/tube; 15 tubes
- Thaw 4 tubes on ice, we will use 100cells per reaction
Bisulfite Conversion
- Proceed with Zymo column based method with improvement tweak protocol
- Elute with 11ul EB Buffer
Heat Denature
- 92C for 30 sec
- Transfer to ice immediately
MDA
30C 60 min, 85C 3 min, 4C hold