title: GSM4141859: 837siSTAT3poolQ-n2_Grade_S41; Homo sapiens; RNA-Seq
library_construction_protocol: Cells were lysed by adding 350 µl RLT buffer (Qiagen) directly on the well and total RNA was extracted using the Rneasy Mini Kit (Qiagen) according to the manufacturers instructions for extraction of total RNA from human cells. RNA-seq libraries were performed using 500 ng mRNA of a non-stranded RNA Seq, massively-parallel mRNA sequencing approach from Illumina (TruSeq RNA Library Preparation Kit v2, Set A; 48 samples, 12 indexes). Specifically we first optimized the ligation step diluting the adapters concentration to increase ligation efficiency (>94%), and finally we reduce the number of PCR cycles (10 cycles) to avoid PCR duplication artefacts as well as primer dimers in the final library product. Libraries were prepared on the automation (Beckman Coulters Biomek FXP workstation). For accurate quantitation of cDNA libraries a fluorometric-based system, the QuantiFluordsDNA System from Promega, was used. The size of final cDNA libraries was determined by using the dsDNA 905 Reagent Kit (Fragment Analyzer Advanced Bioanalytical) exhibiting a sizing of 300 bp in average. Libraries were pooled and sequenced on the Illumina HiSeq 4000 (SE; 1 x 50 bp; 30-35 Mio reads/sample).