Revision as of 00:48, 28 June 2011 by >Alan6017518
N9-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
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
- 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 8ul of nuclease free H2O (aliquot 2ul to perform PCR Analysis)
- Let sit at RT to dissolve the pellet
Nextera Library Construction (Low input protocol)
- In a qPCR tube, mix
- 3ul genomic DNA
- 1ul 1:5 Nextera illumina compatible transposomes (10 μl Nextera enzyme, 20 μl 1XTE, 20 μl 100% glycerol)
- 1ul 5X NExtera HMW buffer
- Incubate at 55C for 5 min
- Add 45ul MM to each reaction
Content
|
Volume per Reaction
|
MM
|
Water |
18 |
198
|
DNA |
5 |
N/A
|
10uM Orange Primer |
1 |
11
|
10uM Blue Primer |
1 |
11
|
KAPA Supermix |
25 |
275
|
Perform Kapa PCR Reaction
95C 3m
95C 10s
58C 30s
72C 3m
plate read
Goto step 2 repeat 34
72C 10m
Hold 15C
PAGE Analysis
Sanger Analysis