title: GSM6971608: HBEC3KT cells, TGFB1, 72hrs, replicate3; Homo sapiens; RNA-Seq
library_construction_protocol: Total RNA was extracted using RLT buffer supplemented with Beta-mercaptoethanol according to the manufacturer's instructions (Qiagen). Isolated RNA was quantified by spectrophotometry, and RNA concentrations were normalized. 1. PolyA-RNA-seq libraries were prepared with KAPA Stranded mRNA-Seq kit (Roche) according to manufacturer's instruction. RNA samples were normalized to 300 ngs input in total 44ul and 6ul of 1:1000 dilution ERCC (mix 1) (Thermo Fisher) was spiked in to each sample. First, poly A RNA was isolated from total RNA using oligo-dT magnetic beads. Purified RNA was then fragmented at 85C for 6 mins, targeting fragments range 250-300bp. Fragmented RNA is reverse transcribed with an incubation of 25C for 10mins, 42C for 15mins and an inactivation step at 70C for 15mins. This was followed by second strand synthesis at 16C, 60mins. Double stranded cDNA fragments were purified using Ampure XP beads (Beckman). The dscDNA were then A-tailed, and ligated with illumina adaptors (IDT). Adaptor-ligated DNA was purified using Ampure XP beads. This is followed by 10 cycles of PCR amplification. The final library was cleaned up using AMpure XP beads. Quantification of libraries were performed using real-time qPCR (Thermo Fisher). Sequencing was performed on Illumina HiSeq4000 platform generating paired end reads of 75bp.