title: GSM6640352: BJAB, parental, untreated, rep3; Homo sapiens; RNA-Seq
library_construction_protocol: 3 – 5 x106 cells were harvested after inhibitor treatment or after adjusting the cells the day before to 0.7 x 106 cells/ml. RNA was isolated with the Qiagen RNeasy Mini Kit (Cat. No.: 74106) or Stratec InviTrap Spin Universal RNA Mini Kit (Cat. No.: 1060100300) following the manufacturer's recommendation. For high quality RNA isolation, the RNA was harvested using Trizol (Cat. No. 15596018, Invitrogen) before further purification with the Qiagen RNeasy Mini Kit (Cat. No.: 74106). Therefore, 1x107 cells were washed once with PBS, resuspended in 600 μl Trizol and incubated at RT for 5 Min. 120 μl Chloroform (Cat. No.: 3313.4, Roth) were added to Trizol and mixed vigorously by hand for 15 s. The samples were incubated at RT for 3 Min before they were centrifuged at full speed for 5 Min at 4°C. To isolate the RNA the upper aqueous phase was transferred to a new tube and the RNA was isolated with the Qiagen RNeasy Mini Kit (Cat. No.: 74106) after addition of the same volume of 70% EtOH by adding the mixture to the column and proceeding according to the manufacturer's recommendations. RNA integrity number (RIN) of Trizol isolated RNA was measured using the Agilent 2100 Bioanalyzer system. RNAs with a RIN value > 7 were selected for mRNA sequencing (poly-A selected). The libraries were prepared using the TruSeq stranded mRNA Sample preparation kit (Illumina), following the instructions of the manufacturer. After a final QC, the libraries were sequenced in a paired-end mode (2x150 bases) in the Novaseq6000 sequencer (Illumina) with a depth of ≥ 30 Million reads per sample. The raw reads and MultiQC sequencing quality report were collected from the Helmholtz Center Munich Genomics Core.