GSM5334975: B20/0647; pTINCR 2 7D L20_3319_S7; Homo sapiens; RNA-Seq
All samples were treated with 1ug/ml of doxycycline at the indicated time points RNA was isolated using Trizol reagent, following manufacturer's protocol Stranded RNA-Seq libraries were prepared by first depleting cytoplasmatic and mitochondrial ribosomal RNAs using the RiboZero Magnetic Gold Kit (Illumina Inc). Then, cDNA from fragmented rRNA-depleted samples was synthesized using reverse transcriptase and random hexamer primers and converted into sequentiable libraries using the TruSeq Stranded Total RNA kit protocol (Illumina Inc.). Double-stranded cDNA were created by removing the RNA template and synthesizing the second strand in the presence of dUTP in place of dTTP. cDNAs were then end-repaired, index adapter-ligated and PCR amplified. Finally, the prepared libraries were quantified by qPCR using KAPA kit as manufacturer's protocol (KAPA Library Quant Kit, KAPA Biosystems). Based on qPCR concentrations, RNA-Seq libraries were loaded on the Illumina NextSeq 550 and sequenced with 2 × 75 bp paired-end reads at an average depth of 100 million paired reads per library