GSM6637903: LNCaP cells, DMSO treatment, 1; Homo sapiens; RNA-Seq
Following treatment (or control), cells were removed from 37C incubator and washed with cold PBS after removal of media. Cells were then harvested with Trizol reagent (Ambion) at 3 mL per 10cm dish following manufacturer protocol. RNA was precipitated with isopropanol and washed with a 75% ethanol solution and dried at room temperature. RNA was resuspended in DEPC-treated water and immediately frozen and stored at -80C. RNA integrity was verified via agarose gel electrophoresis. Total RNA was first evaluated for its quantity, and quality, using Agilent Bioanalyzer 2100. For RNA quality, a RIN number of 10 was achieved. One hundred nanograms of total RNA was used. cDNA library preparation included mRNA purification/enrichment, RNA fragmentation, cDNA synthesis, ligation of index adaptors, and amplification, following the KAPA mRNA Hyper Prep Kit Technical Data Sheet, KR1352 – v4.17 (Roche Corporate). Each resulting indexed library was quantified, and its quality accessed by Qubit and Agilent Bioanalyzer, and multiple libraries pooled in equal molarity. The pooled libraries were then denatured, and neutralized, before loading to NovaSeq 6000 sequencer at 300pM final concentration for 100bp paired-end sequencing (Illumina, Inc.). Approximately 30-40M reads per library were generated. A Phred quality score (Q score) was used to measure the quality of sequencing. More than 90% of the sequencing reads reached Q30 (99.9% base call accuracy).