GSM9021967: 6h_IFNg_before_infection_1; Mus musculus; RNA-Seq
RNA was isolated using the RNeasy Mini Kit (Qiagen), in combination with QIAshredder homogenization columns and the RNase-Free DNase Set (Qiagen), according to the manufacturer's instructions. The quality of the isolated RNA was determined with a Qubit® (1.0) Fluorometer (Life Technologies, California, USA) and a Fragment Analyzer (Agilent, Santa Clara, California, USA). Only those samples with a 260 nm/280 nm ratio between 1.8–2.1 and a 28S/18S ratio within 1.5–2 were further processed. The TruSeq Stranded mRNA (Illumina, Inc, California, USA) was used in the succeeding steps. Briefly, total RNA samples (100-1000 ng) were polyA enriched and then reverse-transcribed into double-stranded cDNA. The cDNA samples was fragmented, end-repaired and adenylated before ligation of TruSeq adapters containing unique dual indices (UDI) for multiplexing. Fragments containing TruSeq adapters on both ends were selectively enriched with PCR.  The quality and quantity of the enriched libraries were validated using Qubit® (1.0) Fluorometer and the Fragment Analyzer (Agilent, Santa Clara, California, USA).  The product is a smear with an average fragment size of approximately 260 bp. The libraries were normalized to 10nM in Tris-Cl 10 mM, pH8.5 with 0.1% Tween 20. Sequencing was performed as single-end 100 bp reads on the Illumina NovaSeq 6000 platform.