GSM4458391: Group_I_rep_4; Mus musculus; RNA-Seq
Total RNA was extracted from the caudal lobes (right lungs) using phenol extraction in combination with the Mirneasy RNA isolation kit (Qiagen, Hilden, Germany). 3 µg BioPM were chosen as representative dose to study gene expression in lungs of OVA/Sal and OVA/OVA mice. In short, murine lung tissue was homogenized by using the bead homogenization (MP Biomedicals, Illkirch, France) in 600 µl Trizol (Thermofisher, Landsmeer, The Netherlands). RNA isolation was carried out according to the manufacturer's instructions. Gene expression profiles in the mouse lungs were obtained by RNAseq. Therefore, libraries for the Illumina NextSeq were generated using the TruSeq Stranded mRNA Library Prep Kit using 400 ng of total RNA as input according to the manufacturer's instructions. Briefly, the mRNA fraction was purified from total RNA by polyA capture, fragmented and subjected to first-strand cDNA synthesis with random hexamers in the presence of Actinomycin D. The second-strand synthesis was performed incorporating dUTP instead of dTTP. Barcoded DNA adapters were ligated to both ends of the double-stranded cDNA and subjected to PCR amplification. Libraries were subsequently validated for fragment size on a QiaXcel (Qiagen) and quantified using RT-qPCR with a Kapa-kit (Roche Sequencing Store). In each sequencing run, 24 libraries were pooled at equimolar concentrations and sequenced using the Illumina NextSeq 500/550 High Output Kit v2.5 (single-end, 75 Cycles)