GSM7943603: URD1; Mus musculus; Bisulfite-Seq
The E17.5 embryonic kidneys were carefully dissected and excised in ice-cold HBSS buffer. Using a surgical scalpel, the kidneys were sliced and chopped into approximately 0.5- to 1-mm pieces on ice. The chopped kidney fragments were then transferred into an HBSS solution supplemented with 1 mg/ml collagenase (11213865001; Sigma-Aldrich) and dispase (D4693-1G; Sigma-Aldrich) and incubated at 37°C for 30 minutes. The cell suspension was filtered through a 40-mm nylon cell strainer (Corning) and washed twice with cold HBSS. Six2 Cre+/tg GFP+ NPCs were isolated by cell flow cytometry-based cell sorting at the Hebrew University Faculty of Medicine Core Facility. For the RRBS analysis, DNA was isolated from FACS-sorted nephron progenitor cells using the Qiagen DNeasy Blood & Tissue Kit (Cat. 69504) following the manufacturer's instructions. Bisulfite conversion of genomic DNA was carried out using the Qiagen Epitect Bisulfite Kit (Cat. 59104), according to the manufacturer's protocol. . RRBS libraries were prepared using 20 ng of DNA and sequenced on a HiSeq 2500 (Illumina) using 100 bp paired-end sequencing. Samples were distributed across multiple lanes during high-throughput sequencing to minimize batch effects.