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.