GSM2057620: db/db glom rep 1; Mus musculus; RNA-Seq
After placing mice under a surgical plane of anesthesia, mice were perfused by insertion of a butterfly needle into the left ventricle. Cold Perfusion solution consisted of 200ul of Dynabeads M-450 Tosylactivated (Life Technologies) diluted in 40 ml of Hank's Balanced Salt Solution (HBSS). After perfusion, kidneys were harvested on ice, minced and chemically digested. Dynabead perfused glomeruli were retreived by magnetic isolation and RNA extracted by Trizol Reagent, purified over PureLink RNA Easy mini columns (Life Tech) The Illumina compatible libraries were prepared using Illumina’s TruSeq RNA Sample Prep kit v2, as per the manufacturer’s protocol. In brief, Poly-A RNA was enriched using Oligo-dT beads. Enriched Poly-A RNA was fragmented to a median size of 150bp using chemical fragmentation and converted into double stranded cDNA. Ends of the double stranded cDNA were polished, 5′-phosphorylated, and 3’-A tailed for ligation of the Y-shaped indexed adapters. Adapter ligated DNA fragments were PCR amplified, quantified and validated by qPCR, and sequenced on Illumina’s HiSeq 2000 in a paired-end read format for 76 cycles.
GEO Accession
GSM2057620