AlanFung:LabNotes/2015/2015-1-22
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Size Selection of LMS (MONOD V5A) BSPP capture on WGBS libraries prepared from cfDNA of normal control and cancer patient
Background
- Kun thinks the results from the experiment Noi did on Illumina’s plasma samples looks promising.
- He thinks we should repeat the same experiments on all our cancer and plasma samples.
- We should have all those WGBS libraries ready, so it would be just a BSPP capturing experiment (we might need to make more probes though).
- Kun asked Noi to help me identify the reagents and design a capturing experiment on all WGBS libraries
- Noi said she had already finished the BSPP capture on WGBS libraries and she will need my help to pool the libraries and do size selection
- Noi will update her wiki so I can follow her work
- Noi used 600ng of WGBS libraries for capture instead of the 400ng for illumina.
- She reduced the probe to target ratio from 200:1 to 125:1due to limited amount of probes.
- She also used MONOD V5A probe set (LMS cluster) and the rest of the protocol are pretty much the same.
- She quantified the library concentration by PAGE quantification, there seems to be a little high variation between different gels
- She asked me to do size selection of 6 pools (8 individual libraries each) of sequencing libraries as noted in her wiki.
- I will have to cut the get in the range between 300-350np (Do not of over 400bp) since she cropped the images to show me.
- There will probably be high background over 400bp, and that is probably ssDNA form when she did more PCR cycles (should have done 1-2 cycle less)
- Use one 5-well TBE gel per one pool of libraries
- The individual sequencing libraries are in the PCR rack on the top shelf of -20C freezer as shown in the wiki.
- Noi already filled in the sequencing spreadsheet
- For Illumina experiment, Noi suggested Li to only pool sequencing libraries and re-purified with 0.7% AMPure beads to concentrate the libraries and remove the remaining primer dimers.
- However, for Li libraries, I got low background of a smear over 400bp.