Revision as of 01:12, 27 August 2012 by >Noi
WGBS library preparation of low input DNA N37 10 tissues samples using KAPA protocol (from Eppendorf) and epMotion
Preparation of enzyme mix for End repair, A-tailing, Ligation, and Methylation adapters
Sample list
Sample IDs
|
Sample code on tubes
|
Well IDs
|
N37-Cerebellum (CRBL) |
N37-1 |
A1
|
N37-Colon |
N37-2 |
B1
|
N37-Frontal lobe (FL) |
N37-3 |
C1
|
N37-Heart |
N37-4 |
D1
|
N37-Small intestine (SI) |
N37-5 |
E1
|
N37-Liver |
N37-6 |
F1
|
N37-Lung |
N37-7 |
G1
|
N37-Skeletal muscle (SM) |
N37-8 |
H1
|
N37-Pancrease |
N37-9 |
A2
|
N37-Stomach |
N37-10 |
B2
|
End repair
Sheared gDNA 200ng in 50ul
Total reaction in
|
70
|
ul
|
DNA |
50 |
ul
|
End repair reaction mix |
20 |
ul
|
KAPA End repair mix
|
1x rxn
|
10x rxn mix
|
10X End repai buffer |
7.00 |
70.00
|
KAPA End repair enzyme |
3.00 |
30.00
|
H2O |
10.00 |
100.00
|
Total |
20.00 |
200.00
|
- Aliquot to column #1: 20ul (1 & 2: 40ul)
- Add 20ul of End-repair reaction mix
- Mix by pipetting 20 times
- Shake at 1000 rpm for 5min (lid on)
- Incubate at 20C for 30min
- Add 120ul of diluted AMPure beads
- Mix at 750rpm for 5min, 25C (lid on)
- Incubate at 25C for 5 min
- Put on magnet and discard spnt
- Wash twice with 140ul 80% EtOH
- Dry the beads
A-tailing
A-tailing reaction mix
|
1x rxn
|
10x rxn mix
|
KAPA 10X A-tailing buffer |
3.00 |
30.00
|
KAPA A-tailing enzyme |
2.00 |
20.00
|
H2O |
25.00 |
250.00
|
Total |
30.00 |
300.00
|
- Aliquot to column #2: 30ul (1 & 2: 60ul)
- Add 30ul of A-tailing reaction mix
- Mix by pipetting 20 times
- Shake at 1000 rpm for 5min (lid on)
- Incubate at 30C for 30min
- Add 90ul of 20%PEG/2.5M NaCl
- Mix at 750rpm for 5min, 25C (lid on)
- Incubate at 25C for 5 min
- Put on magnet and discard spnt
- Wash twice with 140ul 80% EtOH
- Dry the beads
Adapter ligation
Total reaction in
|
50
|
ul
|
Ligation reaction mix |
40 |
ul
|
Diluted Methylation adapters |
10 |
ul
|
Ligation reaction mix
|
1x rxn
|
10x rxn mix
|
KAPA 5X Ligation buffer |
10.00 |
100.00
|
KAPA DNA ligase |
3.00 |
30.00
|
H2O |
27.00 |
270.00
|
Total |
40.00 |
400.00
|
- Aliquot ligation reaction mix to column #3: 40ul (1 & 2: 80ul)
Methylation adapter preparation
Prepare adapter |
0.2ulx 10rxn |
2.00
|
H2O |
9.8ulx10rxn |
98.00
|
Total |
|
100
|
- Aliquot diluted methylation adapter to column #4: 10ul (1 & 2: 20ul)
- Add 40ul of Ligation reaction mix
- Mix by pipetting 10 times
- Add 10ul of diluted Methylation adapters
- Mix by pipetting 10 times
- Shake at 1000 rpm for 5min (lid on)
- Incubate at 20C for 15min
- Add 50ul of 20%PEG/2.5M NaCl
- Mix at 750rpm for 5min, 25C (lid on)
- Incubate at 25C for 5 min
- Put on magnet and discard spnt
- Wash twice with 140ul 80% EtOH
- Dry the beads
- Resuspend adapter ligated DNA with ~23ul EB buffer