GSM2482745: Gray_Boy; Felis catus; RNA-Seq
Total RNA was isolated from primary ASC cultures using the RNeasy Mini Kit (Qiagen, Inc.), RNA quantity and quality were assessed on a NanoDrop spectrophotometer (Thermo Scientific) and the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA), respectively. Transcriptome profiling was performed using a directional, strand-specific mRNA-Seq approach. Briefly, indexed RNA-Seq libraries were prepared from 200 ng total RNA using the KAPA Stranded mRNA-Seq Kit (Kapa Biosystems, Inc.) according to the manufacturer’s standard protocol. Poly-adenylated mRNA was purified from total RNA and ribosomal RNA removed by binding to oligo(dT) beads, which was followed by RNA fragmentation by incubation at 94ºC in the presence of magnesium. Double-stranded cDNA was then generated by random-primed first-strand synthesis and subsequent second strand synthesis in the presence of dUTP for strand marking. The double-stranded cDNA was then 3’-A tailed and indexed, Illumina-compatible adapters were ligated. The libraries were then enriched by high-fidelity PCR amplification (15 cycles) with KAPA HiFi HotStart DNA Polymerase and adapter-specific primers.
GEO Accession
GSM2482745