GSM5416909: s104188-001-016; Homo sapiens; RNA-Seq
Next generation sequencing was performed in RNA samples extracted from n=6 unstimulated; n=8 TGF-β; n=8 TGF-β+BU conditioned HSC. RNA was isolated using the RNAqeous Microkit (Thermo Fisher Scientific) and obtained RNA concentration were determined spectrophotometrically using the Nanodrop 1000 (Isogen Life Science, De Meern, the Netherlands). To assess the quality of the samples, the concentration of the sample was determined using the Fragment Analyzer. The NEBNext Ultra II Directional RNA Library Prep Kit for Illumina was used to process the sample(s). The sample preparation was performed according to the protocol "NEBNext Ultra II Directional RNA Library Prep Kit for Illumina" (NEB #E7760S/L). Briefly, mRNA was isolated from total RNA using the oligo-dT magnetic beads. After fragmentation of the mRNA, a cDNA synthesis was performed. This was used for ligation with the sequencing adapters and PCR amplicification of the resulting product. The quality and yield after sample preparation was measured with the Fragment Analyzer (see appendices). The size of the resulting products was consistent with the expected size distribution (a broad peak between 300-500 bp). Clustering and DNA sequencing using the NovaSeq6000 v1.5 was performed according to manufacturer's protocols. A concentration of 1.1 nM of DNA was used yielding paired end reads (2x 150bp). NovaSeq control software NCS v1.7 was used. Image analysis, base calling, and quality check was performed with the Illumina data analysis pipeline  RTA3.4.4 and Bcl2fastq v2.20.