Noi/NOTES/2014-12-5: Difference between revisions
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>Noi mNo edit summary |
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:- Add 8ul of End repair/dA-tailing rxn mix to each tube in 8-tube strip | :- Add 8ul of End repair/dA-tailing rxn mix to each tube in 8-tube strip | ||
:- Transfer 22ul of ctDNA to each rxn tube. | :- Transfer 22ul of ctDNA to each rxn tube. | ||
:- | :- Mix well (spin down & pulse-vortexing 10X & spin down) | ||
:- 20C, 30min | :- 20C, 30min | ||
:- 65C, 30min, | :- 65C, 30min, | ||
:- Hold 4C | :- Hold 4C | ||
:- Proceed to Adaptor ligation immediately | :- Proceed to Adaptor ligation immediately | ||
== Adaptor ligation == | |||
==== Adaptor ligation reaction mix ==== | |||
* I prepare 10X diluted TruSeq adaptor (conc. after dilution = 1500nM) Index 1-10 by mixing 2ul of 15uM TruSeq adaptor with 18ul H2O. | |||
{| {{table}} border = 1 | |||
| align="center" style="width:200px;background:#f0f0f0;"|'''Components''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''Volume''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''half rxn''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''11.2x rxn''' | |||
|- | |||
| End-repaired/A-tailed DNA||60.00||30.00||0.00 | |||
|- | |||
| Ligation buffer||30.00||15.00||168.00 | |||
|- | |||
| DNA ligase||10.00||5.00||56.00 | |||
|- | |||
| '''Total'''||'''100.00'''||'''50.00'''||'''20ul/rxn''' | |||
|- | |||
| Diluted adaptor (1.5uM)||10.00||5.00|| | |||
|- | |||
| '''Total'''||'''110.00'''||'''55.00'''|| | |||
|} | |||
:- Add 5ul of 1.5uM diluted Truseq adaptor to each tube | |||
::- Final conc. of adaptor in 55ul ligation reaction is ~136nM | |||
:- Add 20ul of ligation reaction mix to each tube | |||
:- Mix well (spin down & pulse-vortexing 10X & spin down) | |||
:- 20C, 15min | |||
:- Continue to SPRI clean up immediately | |||
==== <u>1X AMPure bead purification</u> ==== | |||
* The instruction suggested to use 0.8X of AMPure bead for clean up, but I use 1X of AMPure bead | |||
- Aliquot AMPure bead in 1.5mL tube and leave at RT | |||
:- Add 55ul AMPure (RT) bead Mix by pipetting 10x | |||
:- Incubate at RT for 15min | |||
:- Transfer to sit on magnet for 5min | |||
:- Discard spnt | |||
:- Wash twice with 200ul freshly prepared 80% EtOH | |||
::- After adding 80% EtOH, wait for 30sec (only the 1st tine wash) before pipetting up and down 5X and discard spnt | |||
:- Dry the bead for 5min | |||
:- Resuspend with 21ul EB buffer | |||
:- Transfer eluted adaptor-ligated DNA to new 8-tube strip | |||
:- Continue to bisulfite conversion. Optional: Adaptor-ligated DNA can be stored at -20C before bisulfite conversion | |||
== Bisulfite conversion == | |||
* Bisulfite conversion kit: EZ DNA Methylation-Lightning Kit (column-based method) , cat# D5030 or D5031 | |||
* EZ-96 DNA Methylation-Lightning™ MagPrep (magnetic bead-based method) is another option for large sample set |
Revision as of 10:50, 7 December 2014
WGBS library prep and BSPP capture of cfDNA from Illumina
Aims
- To generate 10 WGBS libraries from ctDNA of colorectal cancer (CRC) patients from Illumina (received from Kansas University)
- To capture WGBS libraries with MONOD v5 (LMS) probe set from Zhang's lab and CRC probe set from Illumina
Sample info
Subject# | Sample_ID | Plasma volume (ul) | Size (bp) by BA | Total Yield (ng) by BA |
KUCC BRCF 011921 | 10.A13.3 | 964 | 187 | 14 |
KUCC BRCF 005881 | 9.B7.36 | 952 | 178 | 13.2 |
KUCC BRCF 007388 | 9.C28.47 | 876 | 173 | 11.9 |
KUCC BRCF 008966 | 9.I19.36 | 882 | 185 | 4.8 |
KUCC BRCF 008282 | 9.G9.25 | 670 | 191 | 9 |
KUCC BRCF 008116 | 9.F20.56 | 924 | 197 | 5.1 |
KUCC BRCF 007855 | 9.H5.58 | 914 | 223 | 8.6 |
KUCC BRCF 008472 | 9.H3.25 | 1,000 | 219 | 4.6 |
KUCC BRCF 008527 | 9.H7.69 | 1,150 | 237 | 11.1 |
KUCC BRCF 008794 | 9.I7.3 | 770 | 249 | 9.57 |
- Each sample has volume ~22ul
- Each sample has different amount of DNA and we want to use up all DNA for WGBS library prep. To make it simple for qPCR monitoring after adaptor ligation and bisulfite conversion, I re-named the sample ID and run based on the input DNA from low to high.
Subject# | Sample_ID | Total Yield (ng)by BA | Zhang lab ID |
KUCC BRCF 008472 | 9.H3.25 | 4.6 | KU_1 |
KUCC BRCF 008966 | 9.I19.36 | 4.8 | KU_2 |
KUCC BRCF 008116 | 9.F20.56 | 5.1 | KU_3 |
KUCC BRCF 007855 | 9.H5.58 | 8.6 | KU_4 |
KUCC BRCF 008282 | 9.G9.25 | 9 | KU_5 |
KUCC BRCF 008794 | 9.I7.3 | 9.57 | KU_6 |
KUCC BRCF 008527 | 9.H7.69 | 11.1 | KU_7 |
KUCC BRCF 007388 | 9.C28.47 | 11.9 | KU_8 |
KUCC BRCF 005881 | 9.B7.36 | 13.2 | KU_9 |
KUCC BRCF 011921 | 10.A13.3 | 14 | KU_10 |
Part I: WGBS library preparation using KAPA Hyper Prep Kit
End repair & dA-tailing
- Note: I will reduce the reaction volume to be half of the instruction and reduce the reagent volume/amount proportionally.
- KAPA Hyper Prep reaction set up (exactly in the instruction):
- - 50ul of fragmented DNA
- - 7ul of End repair & dA-tailing buffer
- - 3ul of End repair & dA-tailing enzyme mix
- - Total reaction: 60ul
End repair & dA-tailing reaction mix
Components | Volume | half rxn | 11.2x rxn |
End repair/dA-tailing buffer | 7.00 | 3.50 | 39.20 |
End repair/dA-tailing enzyme mix | 3.00 | 1.50 | 16.80 |
H2O | 6.00 | 3.00 | |
Total | 10.00 | 8.00 |
Reaction set up
Sample IDs | Zhang lab ID | Volume | End repair/dA-tailing rxn mix | H2O (ul) | Total (ul) |
9.H3.25 | KU_1 | 22.00 | 8 | 0.00 | 30.00 |
9.I19.36 | KU_2 | 22.00 | 8 | 0.00 | 30.00 |
9.F20.56 | KU_3 | 22.00 | 8 | 0.00 | 30.00 |
9.H5.58 | KU_4 | 22.00 | 8 | 0.00 | 30.00 |
9.G9.25 | KU_5 | 22.00 | 8 | 0.00 | 30.00 |
9.I7.3 | KU_6 | 22.00 | 8 | 0.00 | 30.00 |
9.H7.69 | KU_7 | 22.00 | 8 | 0.00 | 30.00 |
9.C28.47 | KU_8 | 22.00 | 8 | 0.00 | 30.00 |
9.B7.36 | KU_9 | 22.00 | 8 | 0.00 | 30.00 |
10.A13.3 | KU_10 | 22.00 | 8 | 0.00 | 30.00 |
NTC | NTC | 0.00 | 8 | 25.00 | 33.00 |
- - Add 8ul of End repair/dA-tailing rxn mix to each tube in 8-tube strip
- - Transfer 22ul of ctDNA to each rxn tube.
- - Mix well (spin down & pulse-vortexing 10X & spin down)
- - 20C, 30min
- - 65C, 30min,
- - Hold 4C
- - Proceed to Adaptor ligation immediately
Adaptor ligation
Adaptor ligation reaction mix
- I prepare 10X diluted TruSeq adaptor (conc. after dilution = 1500nM) Index 1-10 by mixing 2ul of 15uM TruSeq adaptor with 18ul H2O.
Components | Volume | half rxn | 11.2x rxn |
End-repaired/A-tailed DNA | 60.00 | 30.00 | 0.00 |
Ligation buffer | 30.00 | 15.00 | 168.00 |
DNA ligase | 10.00 | 5.00 | 56.00 |
Total | 100.00 | 50.00 | 20ul/rxn |
Diluted adaptor (1.5uM) | 10.00 | 5.00 | |
Total | 110.00 | 55.00 |
- - Add 5ul of 1.5uM diluted Truseq adaptor to each tube
- - Final conc. of adaptor in 55ul ligation reaction is ~136nM
- - Add 20ul of ligation reaction mix to each tube
- - Mix well (spin down & pulse-vortexing 10X & spin down)
- - 20C, 15min
- - Continue to SPRI clean up immediately
1X AMPure bead purification
- The instruction suggested to use 0.8X of AMPure bead for clean up, but I use 1X of AMPure bead
- Aliquot AMPure bead in 1.5mL tube and leave at RT
- - Add 55ul AMPure (RT) bead Mix by pipetting 10x
- - Incubate at RT for 15min
- - Transfer to sit on magnet for 5min
- - Discard spnt
- - Wash twice with 200ul freshly prepared 80% EtOH
- - After adding 80% EtOH, wait for 30sec (only the 1st tine wash) before pipetting up and down 5X and discard spnt
- - Dry the bead for 5min
- - Resuspend with 21ul EB buffer
- - Transfer eluted adaptor-ligated DNA to new 8-tube strip
- - Continue to bisulfite conversion. Optional: Adaptor-ligated DNA can be stored at -20C before bisulfite conversion
Bisulfite conversion
- Bisulfite conversion kit: EZ DNA Methylation-Lightning Kit (column-based method) , cat# D5030 or D5031
- EZ-96 DNA Methylation-Lightning™ MagPrep (magnetic bead-based method) is another option for large sample set