Noi/NOTES/2014-10-8: Difference between revisions
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= Integration of DNA SMART ChIP-Seq protocol with RRBS for DNA methylation assay | = Integration of DNA SMART ChIP-Seq protocol with RRBS for low input DNA methylation assay = | ||
[[noi:DMR220k_LabNotes|'''Link to calendar''']] | [[noi:DMR220k_LabNotes|'''Link to calendar''']] | ||
* 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. | * 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. | ||
[[Media:DNA_SMART_ChIP-Seq_User_Manual_082014.pdf| '''DNA SMART ChIP-Seq kit Manual''']] | [[Media:DNA_SMART_ChIP-Seq_User_Manual_082014.pdf| '''DNA SMART ChIP-Seq kit Manual''']] | ||
== <u>TEST #1</u> == | == <u>TEST #1</u> == |
Revision as of 19:48, 10 October 2014
Integration of DNA SMART ChIP-Seq protocol with RRBS for low input DNA methylation assay
- 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.