Noi/NOTES/2014-12-5: Difference between revisions

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| KUCC BRCF 011921||10.A13.3||14||KU_10
| KUCC BRCF 011921||10.A13.3||14||KU_10
|}
|}
== <span style="color:blue">Part I: WGBS library preparation using KAPA Hyper Prep Kit</span> ==
== 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 ====
{| {{table}} border = 1
| align="center" style="width:240px;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 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||
|}<br>
==== Reaction set up ====
{| {{table}} border = 1
| align="center" style="width:140px;background:#f0f0f0;"|'''Sample IDs'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Zhang lab ID'''
| align="center" style="width:80px;background:#f0f0f0;"|'''Volume'''
| align="center" style="width:80px;background:#f0f0f0;"|'''End repair/dA-tailing rxn mix'''
| align="center" style="width:80px;background:#f0f0f0;"|'''H2O (ul)'''
| align="center" style="width:80px;background:#f0f0f0;"|'''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. 
:- Spin down & mix well by pulse-vortexing 10X & spin down
:- 20C, 30min
:- 65C, 30min,
:- Hold 4C
:- Proceed to Adaptor ligation immediately

Revision as of 10:31, 7 December 2014

WGBS library prep and BSPP capture of cfDNA from Illumina

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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.
- Spin down & mix well by pulse-vortexing 10X & spin down
- 20C, 30min
- 65C, 30min,
- Hold 4C
- Proceed to Adaptor ligation immediately