GSM8447073: Keogenic diet 1; Mus musculus; RNA-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 RNA extraction, 50,000-100,000 cells were washed in cold HBSS, and total RNA was extracted using the Qiagen RNeasy Micro Kit (74004; Qiagen). Libraries were prepared from RNA samples using a SMARTer® Stranded Total RNA-Seq Kit v3 (634485; Takara Bio). The libraries were barcoded and pooled for multiplex sequencing. The pooled DNA library was loaded on a NovaSeq 6000 SP Reagent Kit v1.5 (100 cycles) cartridge (Illumina) and sequenced on an Illumina Novaseq 6000 System using a single-read sequencing condition of 100 cycles.