GSM3900887: HFF, Mock_1; Homo sapiens; RNA-Seq
1 ml TRIzol reagent (Invitrogen) was added to each T25 and the cells were scraped. Lysates were collected into RNAse/DNAse-free Eppendorf tubes and frozen at −20 °C. Total RNA was extracted following the manufacturer's instructions, with some modifications. Briefly: frozen samples were thawed on ice and 0.2 ml chloroform was added to TRIzol suspensions, which were then mixed by inverting 10 times, and incubated for 5 minute. Tubes were then spun at 12,000 rpm for 15 min at 4 °C. RNA in the aqueous phase was transferred into a fresh tube and 0.5 ml absolute isopropyl alcohol was added. Each tube was inverted three times and incubated at 4°C for 10 min. They were then spun at 12,000 rpm for 20 min at 4 °C. After decanting the supernatants, RNA pellets were washed with 1 ml 75% ethanol. Tubes were mixed by inverting 10 times and then spun at 12,000 rpm for 20 min at 4°C. Supernatants were removed and the RNA pellets were air-dried in open tubes for approximately 10 min. The RNA pellets were resuspended in 30 μl RNase-free DEPC-water. Multiplex sequencing libraries were generated with RNA Sample Prep Kit (Illumina) according to manufacturer's instructions and pooled for a single high-throughput sequencing run using the Illumina NextSeq platform (Illumina Nextseq 500 model instrument) at the Stanford University Functional Genomic Facility (SFGF).