GSM9109149: Intestinal epithelial cells, colon, CARD14(E138A) 2; Mus musculus; RNA-Seq
RNA was isolated using an RNeasy Plus micro kit (74134, QIAGEN) according to the manufacturer's instructions. RNA concentration and purity were determined spectrophotometrically using the NanoDrop ND-1000 (NanoDrop Technologies), and RNA integrity was assessed using the Bioanalyzer 2100 (Agilent). Per sample, 250 ng of total RNA was used as input Illumina TruSeq® Stranded mRNA Sample Prep Kit (protocol version: # 1000000040498 v00 October 2017) Poly-A containing mRNA molecules were purified from total RNA using poly-T oligo-attached magnetic beads. mRNA was then reverse-transcribed into first-strand cDNA using random primers, followed by second-strand synthesis using DNA Polymerase I and RNase H to generate double-stranded cDNA. The cDNA fragments were end-repaired and extended with a single 'A' base at the 3' ends. Indexing adapters were ligated to the cDNA fragments, introducing unique barcodes per sample. Enrichment PCR was performed to amplify adapter-ligated fragments. Libraries were equimolarly pooled and sequenced on an Illumina NovaSeq 6000 using a v1.5 kit, S1 flowcell, and single-end read configuration (100-8-8-0) at the VIB Nucleomics Core https://nucleomicscore.sites.vib.be/