GSM2970980: 1st ∆myr1_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. NA samples were submitted to the Stanford University Functional Genomic Facility (SFGF) for purity analysis using the Agilent 2100 Bioanalyzer. 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).