AlanFung:LabNotes/2014/2014-8-14

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Low Input (1ng) WGBS Libraries from Normal Control's Plasma Sample (No DNA Fragmentation Step)[edit]

Sample[edit]

Zhang Id Sample Conc. in the Qubit Unit uL used Dilution Sample Conc. (ng/ul) Volume Yield (ng)
15898001 NC-2 3.25 ng/mL 2 100 0.325 42 13.65
16188001 NC-3 5.38 ng/mL 2 100 0.538 42 22.6

End Repair & A-Tailing[edit]

Zhang Id Sample Sample Conc. (ng/ul) Volume for 1ng (ul) H2O Klenow,exo-:dA:dT:dC:dG Mix Total
15898001 NC-2 0.325 3.08 6.92 10 20
16188001 NC-3 0.538 1.86 8.14 10 20
n/a Neg. Control n/a n/a 10 10 20
Components 1x rxn 3x rxn
10X Tango buffer 2 6
dA:dC:dG (20:2:2mM) 1 3
Klenow fragment, exo- (5U/ul) 1 3
H2O 6 18
Total 10  
- Add H2O to each tube
- Add 1ng of DNA to each tube
- Add 10ul of Klwnow, exo- and dA:dT:dC:dG reaction mix to each tube of DNA. Pipette few times
- Spin down the tube
- Mix by gentle pulse-vortexing on PCR rack 10x
- Spin down the tube
- 30C, 20min --> 37C, 20min --> hold at 4C (no heat lid)
- 75C, 10min --> hold at 4C (heat lid on)

Methylated adaptor ligation[edit]

  • Add 1ul of 1:20 diluted TruSeq adaptors to each end-repaired DNA in total reaction volume 25ul
Exp ID TruSeq Index
NC-2 1
NC-3 2
Neg. Control 4
  • Prepare Ligation reaction mix
Components 1x rxn 3x rxn
dA-tailed DNA 20 0
10X Tango buffer 0.5 1.5
HC T4 DNA ligase (30units/ul) 1 3
10mM ATP 1.25 3.75
H2O 1.25 3.75
Total 24  
- Add 1ul of 1:20 diluted TruSeq adaptors and pipette few times to make sure adaptors were added to the reaction
- Add 4ul of ligation reaction mix 
- Spin down the tube
- Mix by gentle pulse-vortexing on PCR rack 10x
- Spin down the tube
- 16C, 20h (no heat lid)
- Heat inactivate at 65C, 20min --> hold at 4C (heat lid on)
- Continue to bisulfite conversion

Bisulfite Conversion[edit]

Performed bisulfite conversion using EZ DNA Methylation-Gold™ Kit and did the same procedure following manufacturer's instruction and elute with 42ul Elution buffer. This kit is the same kit as LifeTech MethylCode™ Bisulfite Conversion Kit.

Prep

- Prepare 3 tubes of complete CT Conversion Reagent by adding 800ul H2O, 50ul Resuspension Buffer, and 300ul Dilution Buffer
- Mix well and spin down the tube
- Put on thermomixer at 37C for 15min, 1,000rpm
- Solution should be clear
- Spin down the tube
- Add 120ul complete CT Converson Reagent to 30ul bead-purified adaptor ligated DNA --> total volume 150ul
- Mix by pipetting 10X with multi-channel pipette
- Spin down the tube
- Incubate following below program
- 98°C for 10 minutes (DNA denaturation)
- 64°C for 2.5 hours (Bisulfite conversion)
- 4°C storage for up to 20 hours or continue to desulfonation
- Mix 600ul of Binding Buffer with 1ul of 10ng/ul tRNA. For many reaction, make multiple mix.
- Add 601ul Binding Buffer and tRNA mix to each column
- Adjust p200 to 200ul and transfer bisulfite treated DNA to the column
- Mix by pipetting 5x
- Spin down column at 14,000 rpm for 30s
- Discard spnt
- Add 100ul Wash Buffer (Aliquot Wash Buffer in 5mL tube)
- Spin down column at 14,000 rpm for 30s (do not discard spnt)
- Add 200ul Desulfonation Buffer (aliquot Desulfonation Buffer in 2mL or 5mL tube)
- Incubate for 20mins
- Spin down column at 14,000 rpm for 30s (do not discard spnt)
- Add 200ul Wash Buffer
- Spin down column at 14,000 rpm for 30s
- Discard spnt
- Add 200ul Wash Buffer
- Spin down column at 14,000 rpm for 3min (do not discard spnt)
- Transfer column to prepared 1.5mL LoBind tube & make sure that column does not touch spnt in the collecting tube before transferring to new tube
- Add 32ul of Elution Buffer (warm Elution Buffer at 60C for 10min)
- Wait for 2min
- Spin down column at 12,000 rpm for 1min
- Continue to amplification (During elution step, PCR mix should be prepared)

Amplification[edit]

1st round PCR (fix 12 cycles)[edit]

Components Conc unit Final conc./amount unit Volume (ul) 3 rxn mix
Bis-cvt DNA         30  
10X Reaction buffer 10 X 1 X 5 15
dNTP mix 10 mM 0.25 mM 1.25 3.75
TruS_F/R 10 uM 0.3 uM 1.5 4.5
50X SYBG 50 X 0.4 X 0.4 1.2
PfuTurbo Cx 2.5 Unit/ul 1 unit 1 3
H2O         10.85 32.55
Total         50  
- Aliquot 20 + 30ul BIS-DNA template
- Mix well
95C for 2min --> [95C for 20sec -> 60C for 30sec -> 72C for 1min] X 12 or 14X cycles --> 72C for 2min
- Purify with 1.25X AMPure beads
- Elute with 42ul EB Buffer
- Continue to 2nd round PCR. No PAGE verification.

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