GSM5934917: 96h_Purm_100nM_N2 [4535_AF]; Mus musculus; RNA-Seq
For collection of the samples, the medium was aspirated from the wells and cells were washed once with 1 mL PBS. Afterwards 600 µL of RLT buffer (from RNeasy Plus Mini kit, #74134) was added to each well and the cells were collected in RNase free 1.5 mL tubes (supplemented with the RNeasy Plus Mini kit, #74134). For complete lysis, the cells were snap frozen once in liquid nitrogen and thawed on ice. The total RNA was extracted using the RNeasy plus mini kit (including the optional on column DNase I digest) according to the manufacturer's instructions. RNA concentrations and qualities were accessed with the Qubit 4.0 fluorometer, using the QubitTM RNA BR assay kit and the QubitTM RNA IQ assay kit, according to the instructions of the manufacturer. Until further usage, the isolated RNA was snap frozen and stored at -80°C.  RNeasy plus mini kit (Qiagen, #74134)  DNase MAX kit (Qiagen, #15200) RNA quality and quantity was assessed by Agilent Bioanalyser Nanochip. Next, Poly-A RNA was enriched from 500 ng total RNA by the "®Poly(A) mRNA Magnetic Isolation Module" (New England Biolabs) and directional RNA-seq libraries were prepared as described in "Ultra™ II Directional RNA Library Prep Kit for Illumina" (New England Biolabs). Library concentration was enriched by PCR with dual barcoded primers. Concentration of the libraries were determined by Qubit HS kit (Thermo) and the fragment size and quality of libraries screened by Agilent TapeStation. Libraries were then diluted and pooled on a liquid handling station (Biomek i7, Beckman Coulter). Sequencing-by-synthesis was done on an Illumina HiSeq 3000 device at the Max Planck Genome center Cologne in paired-end read mode 2 x 150 bp with a HiSeq 3000 PE Cluster Kit and a HiSeq 3000 SBS Kit (300 cycle). Finally, data was trimmed to 1 x 150 bp.