Noi/NOTES/2014-9-8: Difference between revisions
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| COLO2-03||1.11||22.2 | | COLO2-03||1.11||22.2 | ||
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* 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. | * 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 == | == 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 | * 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 |
Revision as of 17:56, 11 September 2014
Preparation of WGBS libraries of ctDNA/cfDNA from Illumina (no DNA fragmentation step)
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 |
- 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
2nd round PCR (large volume)
- From gel image above, the amplification for sample cf1 and cf4 were still saturated, so I ran 3 cycles for these two samples and 4 cycles for sample cf2 and cf3 in total 170ul (mix before split into 3x of 55ul)
Components | Conc | unit | Final conc. | unit | Volume (ul) | 6 rxn mix |
Purified 1st round DNA | 34.00 | |||||
5X Phusion HF buffer | 5 | X | 1 | X | 34.00 | 204.00 |
dNTP mix | 10 | mM | 0.25 | mM | 4.25 | 25.50 |
TruS_F/R | 10 | uM | 0.3 | uM | 5.10 | 30.60 |
50X SYBR | 50 | X | 0.4 | X | 1.36 | 8.16 |
Phusion HF | 2 | unit/ul | 1.70 | 10.20 | ||
H2O | 89.59 | 537.54 | ||||
Total | 170.00 |
- - Aliquot 136 + 34ul DNA template
- - Mix well. Split 3X of 55ul for each condition
- 98C for 2min --> [98C for 10sec -> 60C for 30sec -> 72C for 1min] X [cf1 &cf4: 3X, cf2 & cf3: 4 X] cycles --> 72C for 2min
- 98C for 2min --> [98C for 10sec -> 60C for 30sec -> 72C for 1min] X [cf1 &cf4: 3X, cf2 & cf3: 4 X] cycles --> 72C for 2min
- - 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