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>Noi (Created page with "= Preparation of WGBS libraries of small fragment DNA in plasma samples (TEST) = '''Calendar''' * Rationale: Will be added * Aim: To generate WGBS li...") |
>Noi mNo edit summary |
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(2 intermediate revisions by the same user not shown) | |||
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| NTC||0.00||0.00||9.80||10.20||20.00 | | NTC||0.00||0.00||9.80||10.20||20.00 | ||
|} | |} | ||
==== Klenow exo- and dA:dG:dC reaction mix ==== | ==== <u>Klenow exo- and dA:dG:dC reaction mix</u> ==== | ||
{| {{table}} border = 1 | {| {{table}} border = 1 | ||
| align="center" style="background:#f0f0f0;"|'''Components''' | | align="center" style="background:#f0f0f0;"|'''Components''' | ||
Line 49: | Line 49: | ||
<br> | <br> | ||
== 2) Methylated adaptor ligation == | == 2) Methylated adaptor ligation == | ||
* Add 1ul of 1:20 diluted TruSeq adaptors to each end-repaired DNA in total reaction volume 25ul | |||
==== <u>Ligation reaction mix</u> ==== | |||
{| {{table}} border = 1 | |||
| align="center" style="background:#f0f0f0;"|'''Components''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''1x rxn''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''3x rxn''' | |||
|- | |||
| dA-tailed DNA||20.00||0.00 | |||
|- | |||
| 10X Tango buffer||0.50||1.50 | |||
|- | |||
| HC T4 DNA ligase (30units/ul)||1.00||3.00 | |||
|- | |||
| 10mM ATP ||1.25||3.75 | |||
|- | |||
| H2O||1.25||3.75 | |||
|- | |||
| Total||24.00|| | |||
|} | |||
:{| {{table}} class = wikitable | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''Sample''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''TruSeq Index''' | |||
|- | |||
| PC-P-1||21 | |||
|- | |||
| PC-P-2||22 | |||
|- | |||
| NTC||22 | |||
|} | |||
:- 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 | |||
== 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. | |||
'''''<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 | |||
:- 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 | |||
:- <u>Discard spnt</u> | |||
:- Add 100ul Wash Buffer (Aliquot Wash Buffer in 5mL tube) | |||
:- Spin down column at 14,000 rpm for 30s <u>(do not discard spnt)</u> | |||
:- Add 200ul Desulfonation Buffer (aliquot Desulfonation Buffer in 2mL or 5mL tube) | |||
:- Spin down column at 14,000 rpm for 30s <u>(do not discard spnt)</u> | |||
:- Add 200ul Wash Buffer | |||
:- Spin down column at 14,000 rpm for 30s | |||
:- <u>Discard spnt</u> | |||
:- Add 200ul Wash Buffer | |||
:- Spin down column at 14,000 rpm for 3min <u>(do not discard spnt)</u> | |||
:- 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 == | |||
* Minor changes: Since 1ng of DNA input was not fragmented even there is a fraction of small fragments, I increase 1st round PCR from 12 cycles to 14 cycles to increase the amplified template. | |||
* To avoid losing small fragments below 200bp, I purified 1st round PCR with 1.25X AMPure bead. I tried not to increase the beads up to 1.5X or 2X since I did not want to get more adaptor dimers contamination in the next round PCR. I should have tested doing single round PCR for ~15-20 cycles and see if I can get sequencing libraries without 2nd round PCR. | |||
=== 1st round PCR (fix 14 cycles) === | |||
{| {{table}} border = 1 | |||
| align="center" style="width:140px;background:#f0f0f0;"|'''Components''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''Conc''' | |||
| align="center" style="width:60px;background:#f0f0f0;"|'''unit''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''Final conc./amount''' | |||
| align="center" style="width:60px;background:#f0f0f0;"|'''unit''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''Volume (ul)''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''4.5 rxn mix''' | |||
|- | |||
| Bis-cvt DNA||||||||||30.00||0.00 | |||
|- | |||
| 10X Reaction buffer||10||X||1||X||5.00||22.50 | |||
|- | |||
| dNTP mix||10||mM||0.25||mM||1.25||5.63 | |||
|- | |||
| TruS_F/R||10||uM||0.3||uM||1.50||6.75 | |||
|- | |||
| 50X SYBG||50||X||0.4||X||0.40||1.80 | |||
|- | |||
| PfuTurbo Cx||2.5||Unit/ul||1||unit||1.00||4.50 | |||
|- | |||
| H2O||||||||||10.85||48.83 | |||
|- | |||
| Total ||||||||||50.00|| | |||
|} | |||
:- Aliquot 20 + 30ul BIS-DNA template | |||
:- Mix well | |||
:: ''95C for 2min --> [95C for 20sec -> 60C for 30sec -> 72C for 1min] X 14 cycles --> 72C for 2min'' | |||
:- Purify with 1.25X AMPure beads (incubate DNA with beads 30min, the rest steps are the same as previous experiment) | |||
:- Elute with 42ul EB Buffer | |||
:- Continue to 2nd round PCR. No PAGE verification. | |||
=== 2nd round PCR === | |||
==== OPTIMIZE PCR cycle number ==== | |||
{| {{table}} border = 1 | |||
| align="center" style="width:140px;background:#f0f0f0;"|'''Components''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''Conc''' | |||
| align="center" style="width:60px;background:#f0f0f0;"|'''unit''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''Final conc.''' | |||
| align="center" style="width:60px;background:#f0f0f0;"|'''unit''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''Volume (ul)''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''17 rxn mix''' | |||
|- | |||
| Purified 1st round DNA||||||||||3.00||0.00 | |||
|- | |||
| 5X Phusion HF buffer||5||X||1||X||2.00||34.00 | |||
|- | |||
| dNTP mix||10||mM||0.25||mM||0.25||4.25 | |||
|- | |||
| TruS_F/R||10||uM||0.3||uM||0.30||5.10 | |||
|- | |||
| 50X SYBR||50||X||0.4||X||0.08||1.36 | |||
|- | |||
| Phusion HF||2||unit/ul||||||0.10||1.70 | |||
|- | |||
| H2O||||||||||4.27||72.59 | |||
|- | |||
| Total||||||||||10.00|| | |||
|} | |||
:- Aliquot 28 + 12ul DNA template | |||
:- Mix well. Split 4X of 9ul | |||
::''98C for 2min --> [98C for 10sec -> '''60C for 30sec''' -> 72C for 1min] X [8X, 9X, 10X, 12X] cycles --> 72C for 2min''<br> | |||
:- Added 3ul 6X loading dye to each strip tube & mix well | |||
:- Load 6ul PCR/dye mix in 6% TBe gel and run at 250 volts for 21 min | |||
* I planned to optimize PCR cycle number between 10-16 cycles by doing PCR in small volume (~10ul). However, based on qPCR curves saturated vary quick at ~8x. so I stopped reaction at 8X, 9X, 10X, and 12X cycles. | |||
* It looked like I should test PCR cycles at lower cycle number. I repeat experiment with the same condition at 6X and 7X cycles. | |||
[[File:ZhangLab_2 2014-08-01_noMspIRRBS_6-7cycles.jpg|460px]] [[File:ZhangLab_2 2014-08-01_noMspIRRBS_8-9cycles.jpg| 500px]] | |||
[[File:ZhangLab_2 2014-08-01_noMspIRRBS_10-12cycles.jpg| 460px]] | |||
1: PC-P-1 | |||
2: PC-P-2 | |||
3: NTC | |||
4: NTC PCR |
Latest revision as of 17:24, 11 September 2014
Preparation of WGBS libraries of small fragment DNA in plasma samples (TEST)[edit]
- Rationale: Will be added
- Aim: To generate WGBS libraries of ctDNA without fragmentation (either enzymatic or shearing) to exclude all large fragment DNA derived from lysed blood cell in sequencing library.
- I chose two samples of PC-P samples which have total DNA more than 10ng to test the protocol
- PC-P-1 (conc. 0.473 ng/ul, total amount 14.19ng)
- PC-P-2 (conc. 0.852 ng/ul, total amount 25.56ng)
1) End-repair/dA-tailing[edit]
- Incubate plasma DNA with 5U Klenow fragment, exo- and dA:dC:dG (final conc. 1:0.1:0.1mM) in total volume 20ul
Sample | Conc. (ng/ul) | Volume for 1ng | H2O | Klenow,exo-:dA:dC:dG Mix | Total |
PC-P-1 | 0.47 | 2.11 | 7.69 | 10.20 | 20.00 |
PC-P-2 | 0.85 | 1.17 | 8.63 | 10.20 | 20.00 |
NTC | 0.00 | 0.00 | 9.80 | 10.20 | 20.00 |
Klenow exo- and dA:dG:dC reaction mix[edit]
Components | 1x rxn | 3.50x rxn |
10X Tango buffer | 2.00 | 7.00 |
dA:dC:dG (20:2:2mM) | 1.00 | 3.50 |
Klenow fragment, exo- (5U/ul) | 1.00 | 3.50 |
H2O | 6.20 | 21.70 |
Total | 10.20 |
- - Add H2O to each tube
- - Add 1ng of DNA to each tube
- - Add 10.2ul of Klwnow, exo- and dA: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)
- No AMPure bead purification
2) Methylated adaptor ligation[edit]
- Add 1ul of 1:20 diluted TruSeq adaptors to each end-repaired DNA in total reaction volume 25ul
Ligation reaction mix[edit]
Components | 1x rxn | 3x rxn |
dA-tailed DNA | 20.00 | 0.00 |
10X Tango buffer | 0.50 | 1.50 |
HC T4 DNA ligase (30units/ul) | 1.00 | 3.00 |
10mM ATP | 1.25 | 3.75 |
H2O | 1.25 | 3.75 |
Total | 24.00 |
Sample TruSeq Index PC-P-1 21 PC-P-2 22 NTC 22
- - 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
4) 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 1 tubes 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[edit]
- Minor changes: Since 1ng of DNA input was not fragmented even there is a fraction of small fragments, I increase 1st round PCR from 12 cycles to 14 cycles to increase the amplified template.
- To avoid losing small fragments below 200bp, I purified 1st round PCR with 1.25X AMPure bead. I tried not to increase the beads up to 1.5X or 2X since I did not want to get more adaptor dimers contamination in the next round PCR. I should have tested doing single round PCR for ~15-20 cycles and see if I can get sequencing libraries without 2nd round PCR.
1st round PCR (fix 14 cycles)[edit]
Components | Conc | unit | Final conc./amount | unit | Volume (ul) | 4.5 rxn mix |
Bis-cvt DNA | 30.00 | 0.00 | ||||
10X Reaction buffer | 10 | X | 1 | X | 5.00 | 22.50 |
dNTP mix | 10 | mM | 0.25 | mM | 1.25 | 5.63 |
TruS_F/R | 10 | uM | 0.3 | uM | 1.50 | 6.75 |
50X SYBG | 50 | X | 0.4 | X | 0.40 | 1.80 |
PfuTurbo Cx | 2.5 | Unit/ul | 1 | unit | 1.00 | 4.50 |
H2O | 10.85 | 48.83 | ||||
Total | 50.00 |
- - Aliquot 20 + 30ul BIS-DNA template
- - Mix well
- 95C for 2min --> [95C for 20sec -> 60C for 30sec -> 72C for 1min] X 14 cycles --> 72C for 2min
- - Purify with 1.25X AMPure beads (incubate DNA with beads 30min, the rest steps are the same as previous experiment)
- - Elute with 42ul EB Buffer
- - Continue to 2nd round PCR. No PAGE verification.
2nd round PCR[edit]
OPTIMIZE PCR cycle number[edit]
Components | Conc | unit | Final conc. | unit | Volume (ul) | 17 rxn mix |
Purified 1st round DNA | 3.00 | 0.00 | ||||
5X Phusion HF buffer | 5 | X | 1 | X | 2.00 | 34.00 |
dNTP mix | 10 | mM | 0.25 | mM | 0.25 | 4.25 |
TruS_F/R | 10 | uM | 0.3 | uM | 0.30 | 5.10 |
50X SYBR | 50 | X | 0.4 | X | 0.08 | 1.36 |
Phusion HF | 2 | unit/ul | 0.10 | 1.70 | ||
H2O | 4.27 | 72.59 | ||||
Total | 10.00 |
- - Aliquot 28 + 12ul DNA template
- - Mix well. Split 4X of 9ul
- 98C for 2min --> [98C for 10sec -> 60C for 30sec -> 72C for 1min] X [8X, 9X, 10X, 12X] cycles --> 72C for 2min
- 98C for 2min --> [98C for 10sec -> 60C for 30sec -> 72C for 1min] X [8X, 9X, 10X, 12X] cycles --> 72C for 2min
- - Added 3ul 6X loading dye to each strip tube & mix well
- - Load 6ul PCR/dye mix in 6% TBe gel and run at 250 volts for 21 min
- I planned to optimize PCR cycle number between 10-16 cycles by doing PCR in small volume (~10ul). However, based on qPCR curves saturated vary quick at ~8x. so I stopped reaction at 8X, 9X, 10X, and 12X cycles.
- It looked like I should test PCR cycles at lower cycle number. I repeat experiment with the same condition at 6X and 7X cycles.
File:ZhangLab 2 2014-08-01 noMspIRRBS 6-7cycles.jpg File:ZhangLab 2 2014-08-01 noMspIRRBS 8-9cycles.jpg File:ZhangLab 2 2014-08-01 noMspIRRBS 10-12cycles.jpg 1: PC-P-1 2: PC-P-2 3: NTC 4: NTC PCR