GSM8390423: 1243_0013_IPF - Fibroblast - TGFb1_PGE2_1uM; Homo sapiens; RNA-Seq
Cells were washed with ice-cold PBS, before RNA lysates were generated using 350 µL/well RLT buffer (Qiagen, Cat.No. 1053393). Generated RNA lysates  were homogenized using QIAshredder homogenizers (QIAGEN, Cat.No. 79656), and further processed for RNA extraction using MagMAX™ total RNA isolation kit (Thermo Fisher Scientific, Cat.No. AM1830) including a DNA digestion step on a KingFisher™ Flex Purification System (Thermo Fisher Scientific) according to manufacturer's instructions. An input of 250 ng was employed for bulk mRNA-seq library preparation with the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs, Cat. No. E7760), together with the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs, Cat. No. E7490) and NEBNext Multiplex Oligos for Illumina (New England Biolabs, Cat. No. E7600) as per the manufacturer's instructions. Ampure XP beads (Beckman Coulter, Cat. No. A63880) for double-stranded cDNA purification were used instead of the recommended SPRIselect Beads. Libraries were amplified with 13 PCR cycles. The final mRNA-seq libraries were eluted in 40 µL EB Buffer (Qiagen, Cat. No. 19086) and were quantified by the High Sensitivity dsDNA Quant-iT Assay Kit (Thermo Fisher Scientific, Cat. No. Q33120) on a Synergy HTX Multimode Reader (BioTek). Libraries were assessed for size distribution and adapter dimer presence (<0.5 %) by the High Sensitivity Small Fragment Kit on a 96-channel Fragment Analyzer (Agilent, Cat. No. DNF-477-0500). Libraries were normalized on the MicroLab STAR (Hamilton) and pooled. Libraries were sequenced on a NovaSeq 6000 (Illumina) with dual index, paired-end reads (Read Parameter: Rd1:101, Rd2:10, Rd3:10, Rd4:101) with an average sequencing depth of ~>30 million Pass-Filter reads per sample.