Revision as of 23:39, 29 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)
- C1-600pg GM12878 gDNA
- C2-600pg GM12878 gDNA>Noi
- C3-600pg GM12878 gDNA>Noi
- 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
- Preparation of Proteinase K
- Add 260 µl (D5020) of Proteinase K Storage Buffer to the tube containing Proteinase K. Dissolve completely
- 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
- Spin down tubes and add 9ul nuclease free water and transfer cells to 0.2ml tubes
Content
|
Volume per Reaction
|
M-Digestion Buffer (2X) |
10
|
Sample |
9
|
Proteinase K |
1
|
Total |
20
|
- Incubate the samples at 50C for 20min
Bisulfite Conversion
- Dilute GM12878 gDNA from 50ng/ul to 0.5ng/ul (1:100)
- Setup two more 600pg GM12878 gDNA reactions for Noi to perform capture using the 330k probe set
0.5ng/ul GM12878 gDNA
|
Volume Per Reaction
|
Make up to 20ul with H2O
|
600pg |
1.2 |
18.8
|
1.2ng |
2.4 |
17.6
|
- Add 130ul CT Conversion Reagent to all samples
- Perform reaction in thermocycler
- Step1 98C, 8m
- Step2 64C, 3.5hr
- Step3 4C, storage for up to 20 hr
- 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 (CONTINUE ON 6/29)
- 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
- Setup PCR Reaction with Klenow Exo- but without primers
Content
|
Volume Per Reaction
|
MM
|
Water |
17.5 |
154
|
DNA |
5 |
Do not add to MM
|
KAPA Supermix |
25 |
220
|
Klenow Exo Minus |
0.5 |
4.4
|
10uM Orange Primer |
1 |
Do not add to MM
|
10uM Blue Primer |
1 |
Do not add to MM
|
Total |
50 |
378.4
|
- Add 43ul MM to each reaction
- Incubate reactions at RT for 5 min
- Put the tubes back on ice
- Add Primers
- Perform thermal cycling
Perform Kapa PCR Reaction
95C 3m
95C 10s
58C 30s
72C 3m
plate read
Goto step 2 repeat 34
72C 10m
Hold 15C
File:ZhangLab 2011-06-29 1.JPG
- A1-100 sperm cells
- A2-100 sperm cells (repeat)
- B1-100 ESCs
- B2-100 ESCs (repeat)
- C1-600pg GM12878 gDNA
- D-1.2ng GM12878 gDNA
- E1-NTC
- E2-NTC(MDA)
PAGE Analysis
File:ZhangLab 2 2011-06-29 16hr 00min.jpg
Results
- E1-NTC was amplified and clear bands showed up on the gel image, high possibility of contanmination
- E2-NTC added during MDA did not amplify during PCR that shows no contamination was introduced during MDA
To do
- Size Selection on all samples (use clone well gel to extract small amount of sample to do cloning)
- Do cloning on all samples
- Repeat experiment with only gDNA
- gDNA without proteinase k
- gDNA with proteinase k
- gDNA with protease
- NTC but with proteinase K
- NTC without proteinase K
- gDNA with 0.1ul proteinase K
- Run PCR on 5,8 and 9 with bisulfite verification primers
- Perform 5 cycles of PCR to add illumina adapter and barcodes
Sanger Sequencing