Revision as of 20:53, 10 October 2014 by >Noi
Integration of DNA SMART ChIP-Seq protocol with RRBS for low input DNA methylation assay
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- Dr. Zhang suggested to try template switching protocol in stead of the standard adaptor ligation base method to generate bisulfite sequencing library. The idea is to fragment DNA by MspI to enrich only the targets for RRBS then perform bisulfite conversion on MspI-digested DNA. The single strand bisulfite converted DNA will be used as the template for the subsequent steps, including dephosphorylation, poly dT-tailng, first strand cDNA synthesis, and template switching to append the partial adaptor sequence to the newly synthesized cDNA. Then PCR with adaptor primers will be performed to amplified the libraries. One advantage of performing bisulfite conversion before generating sequencing library is that the template with ligated adaptors won't be damaged within the molecules as occurs in standard WGBS protocol.
DNA SMART ChIP-Seq kit Manual
TEST #1
- I used gDNA sample of cancer cell line CANC1 (eq. to XXXX) for the first TEST experiment before moving to gDNA from solid tumor tissue.
- Vary amount of template at 1ng, 100pg and 10pg.
- - Dilute 86.4ng/ul to 2ng/ul --> re-quantify conc. by Qubit: 1.57ng/ul
- - Mix 2.32ul of 86.4ng/ul of CANC1 and 97.68ul of TE buffer
- - Dilute to : 100pg/ul, 10pg/ul, 1pg/ul to used 10ul of each concentration for MspI digestion
MspI reaction Mix
Components
|
1x rxn
|
4.5x rxn
|
MspI (10U/ul) |
1.00 |
4.50
|
10x Tango Buffer |
2.00 |
9.00
|
H2O |
7.00 |
31.50
|
Total |
10.00 |
|
MspI digestion set up
Input amount
|
Stock used (pg/ul)
|
Volume of stock (ul)
|
MspI reaction mix
|
Total (ul)
|
1ng |
100.00 |
10.00 |
10.00 |
20.00
|
100pg |
10.00 |
10.00 |
10.00 |
20.00
|
10pg |
1.00 |
10.00 |
10.00 |
20.00
|
NTC |
|