title: GSM6068447: mur8h1C5 Placebo; Mus musculus; RNA-Seq
library_construction_protocol: Cells were scraped off from the well surface and resuspended in RLT lysis buffer (Qiagen). Total RNA was extracted from these lysed cells using RNeasy micro columns (Qiagen). RNA concentration and purity were determined spectrophotometrically using the Nanodrop ND-8000 (Nanodrop Technologies) and RNA integrity was assessed using a Bioanalyzer 2100 or Fragment Analyzer (Agilent). Per sample, an amount of 100 ng of total RNA was used as input. Using the Illumina TruSeq(r) Stranded mRNA Sample Prep Kit (protocol version: Part # 1000000040498 v00 October 2017) poly-A containing mRNA molecules were purified from the total RNA input using poly-T oligo-attached magnetic beads. In a reverse transcription reaction using random primers, RNA was converted into first strand cDNA and subsequently converted into double-stranded cDNA in a second strand cDNA synthesis reaction using DNA PolymeraseI and RNAse H. The cDNA fragments were extended with a single 'A' base to the 3' ends of the blunt-ended cDNA fragments after which multiple indexing adapters were ligated introducing different barcodes for each sample. Finally, enrichment PCR was carried out to enrich those DNA fragments that have adapter molecules on both ends and to amplify the amount of DNA in the library. Sequence-libraries of each sample were equimolarly pooled and sequenced on Illumina NovaSeq 6000 (S1 flowcell 100 cycles kit v1.5, 100 bp, Single Reads) at the VIB Nucleomics core (www.nucleomics.be).