GSM8459411: ileum, D0, PBS control 3; Mus musculus; RNA-Seq
Approximately 2cm of terminal ileum was dissected and submerged in Tissue protect reagent (Qiagen), and kept at -80C. RNA was extracted using  RNeasy Plus Universal mini kit (Qiagen #73404) and RNA concentration was measured and stored at -80C until ready for sequencing. mRNA was purified from approximately 1000 ng of total RNA with oligo-dT beads and sheared by incubation at 94°C in the presence of Mg2+ (Kapa mRNA HyperPrep).  Following first-strand synthesis with random primers, second strand synthesis and A-tailing were performed with dUTP for generating strand-specific sequencing libraries. Adapters containing 3' dTMP overhangs were ligated to library insert fragments, and library amplification was used to select and amplify fragments carrying the appropriate adapter sequences at both ends. Strands marked with dUTP are not amplified. Indexed libraries that meet appropriate cut-offs for both were quantified by qRT-PCR using a commercially available kit (KAPA Biosystems) and insert size distribution determined with the LabChip GX or Agilent TapeStation. Samples with a yield of ≥0.5 ng/ul were used for sequencing. Sample concentrations were normalized to 2.0 nM and loaded onto an Illumina NovaSeq flow cell at a concentration that yields 30 million post-filtering clusters per sample. Samples were sequenced using 100bp paired-end sequencing according to Illumina protocols.