CUT&RUN in the chromatin landscape of healthy and injured cell types in the human kidney

*** To access this data, please go to Kidney Precision Medicine Project (KPMP) 

CUT&RUN of human kidney nephrectomy tissue using 66,000-200,000 cells/reaction. H3K4me3 (1 biological replicate, with 3 technical replicates). H3K27ac (4 biological replicates, one with 3 technical replicates).  H3K4me1 (1 biological replicate, with 3 technical replicates). H3K27me3 (6 biological replicates, one with 3 technical replicates, and two with isogenic replicates). Liquid nitrogen or OCT-embedded frozen kidney tissue from 6 donors was processed according to the protocol from Epicypher (https://www.epicypher.com/content/documents/protocols/cutana-cut&run-protocol.pdf) with modification. The full modified protocol is published on protocols.io and available here: https://www.protocols.io/view/cut-amp-run-chromatin-profiling-of-human-kidney-ti-bp2l615o1vqe/v1. Antibodies used in this study for CUT&RUN reactions: H3K27ac (Cell Signaling, 8173), H3K27me3 (Cell Signaling, 9733), H3K4me1 (Cell Signaling, 5326), H3K4me3 (Cell Signaling, 9751), and IgG (Cell Signaling, 2729). Up to 3ng of DNA from KPMP donor samples (21-020, 20-687, 20-688) was used to prepare sequencing libraries using the Ion Plus Fragment Library Kit (Thermo Fisher Scientific, 4471252), following manufacturer’s instructions, using 17 cycles to amplify the library. Up to 0.5ng of DNA from KTRC donor samples (3399, 3409, 3447) was used to prepare sequencing libraries using NEBNext Ultra II Library Kit (NEB, E7645), following manufacturer’s instructions, using 18 cycles to amplify the library. Sequencing: 100pM KPMP donor libraries were sequenced on the Ion Torrent Proton to a sequencing depth of 30 million reads (2-3X genome coverage). 0.8 pm KTRC donor libraries were sequenced on the NovaSeq 6000 (Illumina) targeting 100 million paired-end reads. Detailed bioinformatic analysis with command line examples for Illumina sequences can be found here: https://www.protocols.io/view/cut-amp-run-chromatin-profiling-of-human-kidney-ti-bp2l615o1vqe/v1?step=36. Briefly, trimmed fastq files were aligned to the hg38 reference genome using Bowtie2 (for Illumina sequenced) or TMAP (for Ion Torrent sequenced). Aligned reads were extracted and converted to a sorted bam file using SAMtools. Scaled bigWig files were generated using Deeptools, and peaks were called using Macs2.

Published in: DOI: 10.1038/s41467-023-44467-6