GSM8268412: HaCaT cells treated with 10 µM AZD4547 for 5 h, third replicate; Homo sapiens; RNA-Seq
Cells were washed with PBS one time, then total RNA from was isolated using the IBI Scientific RNA isolation kit (IBI Scientific, Dubuque, IO; IB47303) following the manufacturer`s protocol. The quality of the isolated RNA was determined with a Fragment Analyzer (Agilent, Santa Clara, California, USA). Only those samples with a 260 nm/280 nm ratio between 1.8–2.1 and a 28S/18S ratio within 1.5–2 were further processed. The TruSeq Stranded mRNA (Illumina, Inc, California, USA) was used in the succeeding steps. Briefly, total RNA samples (100-1000 ng) were poly A enriched and then reverse-transcribed into double-stranded cDNA. The cDNA samples were fragmented, end-repaired and adenylated before ligation of TruSeq adapters containing unique dual indices (UDI) for multiplexing. Fragments containing TruSeq adapters on both ends were selectively enriched with PCR.  The quality and quantity of the enriched libraries were validated using the Fragment Analyzer (Agilent, Santa Clara, California, USA).  The product is a smear with an average fragment size of approximately 260 bp. The libraries were normalized to 10nM in Tris-Cl 10 mM, pH8.5 with 0.1% Tween 20. Cluster Generation and Sequencing: The Novaseq 6000 (Illumina, Inc, California, USA) was used for cluster generation and sequencing according to standard protocol.  Sequencing were paired end at 2 X150 bp or single end 100 bp.