Noi/NOTES/2014-9-8: Difference between revisions

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* 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.
* 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.
'''''<span style="color:crimson"><u>Prep</u></span>'''''<br>
'''''<span style="color:crimson"><u>Prep</u></span>'''''<br>
:- Prepare 1 tubes of complete CT Conversion Reagent by adding 850ul H2O, 50ul Resuspension Buffer, and 300ul Dilution Buffer
:- Prepare 1 tube of complete CT Conversion Reagent by adding 850ul H2O, 50ul Resuspension Buffer, and 300ul Dilution Buffer
:- Mix well and spin down the tube
:- Mix well and spin down the tube
:- Put on thermomixer at 37C for 15min, 1000rpm
:- Put on thermomixer at 37C for 15min, 1000rpm

Revision as of 17:45, 11 September 2014

Preparation of WGBS libraries of ctDNA/cfDNA from Illumina (no DNA fragmentation step)

Calendar
Sample info.

Re-quantification of cfDNA by Qubit dsDNA HS assay

Sample Conc. By Qubit (ng/ul) Amount (ng)
cfDNA 7.09 49.63
NORM1-02 P1 1.66 33.2
NORM1-03 P1 0.77 15.3
COLO2-03 1.11 22.2
  • One sample from Xuyu (received 2014-08-12). I added more TE buffer and re-quantified concentration (7.09ng/ul). The sample was labeled cdDNA without additional further information.
  • Note that Alan has taken 10ng of each sample for WGBS library prep . I have no time to wait until the library prep done, so I perform experiment in parallel using 5ng of input DNA to make sure that I can continue BSPP capture and get the library ready for Illumina by this Friday.

1) End-repair/dA-tailing

  • Incubate plasma DNA with 5U Klenow fragment, exo- and dA:dC:dG:dT (final conc. 1:0.1:0.1:0.1mM) in total volume 20ul
Zhang lab ID Conc. (ng/ul) Volume for 5ng Exp ID H2O Klenow,exo-:dAdCdG Mix Total Exp ID
cfDNA 7.09 0.71 cf1 9.29 10.00 20.00 cf1
NORM1-02 P1 1.66 3.01 cf2 6.99 10.00 20.00 cf2
NORM1-03 P1 0.77 6.49 cf3 3.51 10.00 20.00 cf3
COLO2-03 1.11 4.50 cf4 5.50 10.00 20.00 cf4
NTC 0.00 0.00 NTC 10.00 10.00 20.00 NTC

Klenow exo- and dA:dG:dC:dT reaction mix

Components 1x rxn 5.5x rxn
10X Tango buffer 2.00 11.00
dA:dC:dG:dT (20:2:2:2mM) 1.00 5.50
Klenow fragment, exo- (5U/ul) 1.00 5.50
H2O 6.00 33.00
Total 10.00
- Add H2O to each tube
- Add 5ng of DNA to each tube
- Add 10ul of Klwnow, exo- and dA:dC:dG:dT 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)
  • No AMPure bead purification


2) Methylated adaptor ligation

  • Add 1ul of 1:20 diluted TruSeq adaptors to each end-repaired DNA in total reaction volume 25ul

Ligation reaction mix

Components 1x rxn 5.5x rxn
dA-tailed DNA 20.00 0.00
10X Tango buffer 0.50 2.50
HC T4 DNA ligase (30units/ul) 1.00 5.00
10mM ATP 1.25 6.25
H2O 1.25 6.25
Total 24.00
Exp ID Sample ID TruSeq Index
cf1 cfDNA 1
cf2 NORM1-02 P1 2
cf3 NORM1-03 P1 4
cf4 COLO2-03 5
NTC NTC 6
- 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, 18h (no heat lid)
- Heat inactivate at 65C, 20min --> hold at 4C (heat lid on)
- Continue to bisulfite conversion

4) Bisulfite conversion

  • 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 1 tube of complete CT Conversion Reagent by adding 850ul H2O, 50ul Resuspension Buffer, and 300ul Dilution Buffer
- Mix well and spin down the tube
- Put on thermomixer at 37C for 15min, 1000rpm
- Solution should be clear
- Spin down the tube
- Add 125ul 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
- Add 601ul Binding Buffer to each column (with 10ng of tRNA in each 601ul Binding Buffer)
- 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)
- 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 30ul 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

  • In previous experiment, I worried about losing small ligated DNA ~200bp, so I used 1.25X AMPure beads to purify 1st round PCR. In this library prep, I want to generate WGBS library for BSPP capture. I want the inser size at least 200bp for capture, so I can remove all the ligate DNA below 200bp by using 1X AMPure beads.

1st round PCR (fix 12 cycles)

  • WILL BE ADD
File:ZhangLab 2 2014-09-10 12hr 45min 1stPCR cfDNA1-4 12cycles 2ulPCR.jpg

2nd round PCR (fix 12 cycles)

OPTIMIZE PCR cycle number

File:ZhangLab 2 2014-09-10 13hr 19min 2ndPCR cfDNA 6-7cycles.jpg
  • It seemed to be too saturated for 6 and 7 cycles. I the tried 4 and 5 cycles and also reduce 1st DNA template in PCR reaction from 30% to 20%
File:ZhangLab 2014-09-10 2ndPCR cfDNA 4-5cycles 2ul-in-10.jpg
  • Perform one round of 1x volume AMPure bead purification because the intensity of adaptor dimer wwas not very high and elute in ~65ul EB buffer
  • Verify libraries by loading 1ul in 6% TBE gel. The 4 samples of 6-P-2 - 6-P-5 WGBS libraries that will be included for BSPP capture were also verified.
File:ZhangLab 2 2014-09-11 01hr 14min PAGE-verify cf1-4 6P-2-5.jpg