Technologies

Epigenetics

CUT&RUN and DNA methylation sequencing: goals, protocols, and metadata standards.

KPMP Epigenetics technologies

CUT&RUN

Goal: To obtain genome-wide profiles of histone post-translational modifications that define chromatin states of healthy and injured/repairing kidney tissue using Cleavage Under Targets and Release Using Nuclease (CUT&RUN). This technology enables the interrogation of chromatin profiles with limited tissue, such as kidney biopsies. 

CUT&RUN workflow: a biopsy fragment is thawed, disaggregated and counted; cells are immobilized, bound with primary antibody and digested with pAG-MNase; released chromatin is sequenced. Protocols, raw and processed files and called peaks go to the KPMP data lake.

‍PROTOCOLS:

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DNA methylation sequencing

Goal: The goal of this technology is to uncover methylation signatures of kidney sub-segments that are key determinants of kidney disease progression. This work will result in a comprehensive set of methylation patterns and genotypes to map back to a kidney atlas. This will allow the characterization of methylation signatures of over 20 million CpG sites in individual kidney compartments from both nephrectomies and biopsies using laser microdissection (LMD) methodology with downstream whole-genome bisulfite-sequencing (WGBS).

Whole-genome bisulfite sequencing workflow: 1) cryosectioning, 2) laser microdissection, 3) DNA isolation, bisulfite conversion and sequencing, 4) alignment with Bismark, 5) analysis in R, 6) upload to the KPMP data lake.

Workflow: 1) Cryosectioning, 2) LMD processing, 3) DNA isolation, bisulfate conversion, WGBS-sequencing, 4) aligning with the reference genome with Bismark, 5) data analysis in R, and genome program visualization, 6) raw and analyzed data are uploaded to the KPMP data lake.

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PROTOCOLS:

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Metadata standards

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