title: GSM3004854: mtHKE3 cells 120 mins post stimulation with TGF-alpha - Replicate 1; Homo sapiens; RNA-Seq
library_construction_protocol: RNA was isolated from the cell lines at 0, 15, 30, 60, 90 and 120 mins post-stimulation using TRIzol (Thermo Fisher Scientific) according to the manufacturer's instructions. RNA was extracted from 3 biological replicates at each timepoint. An ethanol precipitation was used to further purify the RNA. Briefly, 0.1x final volume of 3 M sodium acetate, 20 µg glycogen (Life Technologies) and 2.5X 100% ethanol was used to resuspend RNA. RNA was precipitated at -80ᵒC for a minimum of 2 hours. The precipitant was spun at 13,000 x g for 30 min at 4 ᵒC. The supernatant was removed and the pellet was washed with twice with 75% ethanol, before resuspending in a final volume of 50 µL of RNase-free water. All RNA samples were treated with DNase (Ambion) to remove any contaminating DNA from the purified RNA. Briefly, 2 Units/µL of rDNase I enzyme was added to RNA in 10x DNase I Buffer and incubated at 37ᵒC for 30 min. The reaction was inactivated by addition of DNase Inactivation Reagent and purified DNA-free RNA was extracted from the resultant supernatant. RNA concentration was determined by spectrophotometry on the NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific) and by Qubit assay (Thermo Fisher Scientific). RNA integrity was assessed using a Bioanalyzer 2100 (Agilent). Total RNA was converted to strand-specific Illumina compatible sequencing libraries using the NEXTflex Rapid Directional mRNA-Seq library Kit from BIOO Scientific (Austin, Texas) as per the manufacturer's instructions (v14.10), by staff at the SAHMRI David R. Gunn Genomics facility. Briefly, 200ng of total RNA was polyA selected and the mRNA chemically fragmented prior to reverse transcription and second strand cDNA synthesis using dUTP. The resultant cDNA was poly adenylated before the ligation of Illumina-compatible barcoded sequencing adapters. The cDNA libraries were treated with UDG to degrade the second strand and PCR amplified for 15 cycles prior to assessment using a TapeStation 2200 (Agilent) for quality and Qubit fluorescence assay for quantification. In total, 36 cDNA libraries (2 cell lines X 6 time-points X 3 replicates per timepoint) were generated for sequencing.
attributes.tag: GEO Accession
attributes.value: GSM3004854