Noi/NOTES/2012-8-26

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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

  • Total 30ul/reaction
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

qPCR to check if the end repair, A-tailing, and adapter ligation works

Components 1rxn 11.5 rxn mix
Adapter ligated DNA 1.00 0.00
10uM PCR_F 0.50 5.75
10uM PCR_R.Ind2 0.50 5.75
2x iQ MM 12.50 143.75
H2O 10.50 120.75
Total 25.00 276.00
  • Aliquot 24ul, add 1ul of adapter ligated DNA
Program	
98C 2min --> (98C 15sec -> 64C 20sec -> 72C 30sec) x 12 cycles --> 72C 3min -->  15C Hold
File:20120826 testqPCR adapterligated N37.png