AlanFung:LabNotes/CTCF/2011-6-28: Difference between revisions
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*Spin down at 10,000rpm at 4C for 15min | *Spin down at 10,000rpm at 4C for 15min | ||
*Remove all liquid | *Remove all liquid | ||
*Add 500ul | *Add 500ul 4C chilled 75% ethanol | ||
*Spin at 10,000rpm at 4C for 5 min | *Spin at 10,000rpm at 4C for 5 min | ||
*Remove all liquid | *Remove all liquid |
Revision as of 00:32, 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
- Label tubes A1, A2, B1, B2
- Sperms: ~50cell/tube; 15 tubes
- ES cells: ~50cell/tube; 15 tubes
- Thaw 4 tubes on ice, we will use 100cells per reaction
Bisulfite Conversion
- Setup two more 600pg GM12878 gDNA reactions for Noi to perform capture using the 330k probe set
- Proceed with Zymo column based method with improvement tweak protocol
- Elute with 11ul EB Buffer
- Transfer bisulfite converted samples to 0.2ml tubes for MDA
Heat Denature
- 92C for 30 sec
- Transfer to ice immediately
MDA
- Prepare MDA Master Mix
Content | Volume Per Reaction | MM |
10X RepliPhi Buffer | 2 | 22 |
1mM N9 Primer | 1 | 11 |
25mM dNTP | 0.8 | 8.8 |
RepliPhi Phi29 (100U/ul) | 1 | 11 |
Klenow Exo Minus (10U/ul) | 0.5 | 5.5 |
H2O | 4.7 | 51.7 |
Total | 10 | 110 |
- Add 10ul of MDA mastermix to each sample
Plate Layout | Plate Layout | Plate Layout |
1 | 2 | |
A | A1-100 Sperm Cells | Noi-600pg GM12878 |
B | A2-100 Sperm Cells | Noi-600pg GM12878 |
C | B1-100ESCs | Empty |
D | B2-100ESCs | Empty |
E | C-600pg GM12878 | Empty |
F | D-1.2ng GM12878 | Empty |
G | E1-NTC | Empty |
H | E2-NTC(MDA) | Empty |
Plate Layout | Plate Layout | Plate Layout |
30C 60 min, 85C 3 min, 4C hold
Ethanol Precipitation
Ethanol Precipitation
- Add 2.5X 100% Ethanol
- Add 0.1X 3M NaOAC
- Add 1ul glycoblue
- Store at -80C for 30min
- Spin down at 10,000rpm at 4C for 15min
- Remove all liquid
- Add 500ul 4C chilled 75% ethanol
- Spin at 10,000rpm at 4C for 5 min
- Remove all liquid
- Let dry in hood for 5 min
- Add 6ul of nuclease free H2O
- Let sit at RT to dissolve the pellet