GSE195443
Multimodal single cell sequencing of human diabetic kidney disease implicates chromatin accessibility and genetic background in disease progression [cut-and-run and bulk ATAC-seq to support single cell data]
Purpose: Identification of glucocorticoid recepor binding sites on human kidney proximal tubular cells to better understand epigenetic alterations  in diabetic kidney diseases Methods: CUT&RUN assay libraries for cultured cells or human kidneys were generated with the CUTANA kit (EpiCypher, 14-1048). 500,000 cells were mixed and incubated with Concanavalin A (ConA) conjugated paramagnetic beads. Antibodies to the glucocorticoid receptor were added to target samples and IgG was added to negative controls. The remaining steps were performed according to manufacturer's instructions. Library preparation was performed using the NEBNext Ultra II DNA Library Prep Kit for Illumina (New England BioLabs, E7645S) with manufacturer's instructions, including minor modifications indicated by CUTANA described above. CUT&RUN libraries were sequenced on a NovaSeq instrument (Illumina, 150 bp paired-end reads). For bulk ATAC-seq, 50,000 nuclei were transposed in 25 µL of ATAC-seq transposition mix [12.5 µL 2× Illumina Tagment DNA (TD) buffer; 10.5 µL nuclease-free water; 2.0 µL Tn5 transposase (Illumina/Nextera, FC-121-1030)] and incubated at 37°C for 1 h on a thermomixer. The transposed DNA was purified with QIAGEN MinElute kit (28004) and amplified with dual indexes. Overall design: Cut-and-run for glucocorticoid receptor binding sites on normal human kidney cortex or immortalized renal proximal tubule epithelial cells (hTERT-RPTEC, ATCC). Bulk ATAC-seq on primary RPTEC (Lonza) or hTERT-RPTEC.