GSM5289949: Colorectal Cancer Cells - HCT116_sPDL1_24h_rep1; Homo sapiens; RNA-Seq
RNA was extracted using the Qiagen RNeasy Mini Kit according to manufacture protocol Total RNA was quantified using the Qubit fluorometer (ThermoFisher) with Qubit™ RNA HS Assay Kit (ThermoFisher). Polyadenylated RNA libraries were prepared using QIAseq Stranded mRNA Select Kit (Qiagen). The mRNA Enrichment was obtained starting from 1ug of total RNA using Oligo-dT probes attached to the surface of magnetic beads. Fragmentation was then performed at the reaction temperature of 95°C to obtain an insert size of ~350 bp. Double-stranded cDNA was achieved by a reaction with Rnase H-Reverse Transcriptase in combination with random primers followed by a second-strand synthesis, end repair, and A-addition step. The dual index technology was used to sequence multiple samples thanks to the ligation of Illumina-compatible adapters. Finally, the last amplification step was performed on the purified libraries using the CleanStart PCR Mix, also able to degrade contaminating material. QIAseq Beads were used for reaction cleanups between protocol steps. The quality control and quantification of libraries were performed using the Agilent 2100 Bioanalyzer instrument and High Sensitivity DNA Kit (Agilent). Each QIAseq Stranded library was diluted to a concentration of 4 nM. Then, the libraries with different sample indexes were pooled in equimolar amounts. The pooled libraries at concentration of 1.6 pM were sequenced on Illumina NGS NextSeq 500 platform using 2x 150 bp paired-end mid-output sequencing kit (Illumina).