title: GSM5856709: non_vaccinated_non_inoculated_01; Mesocricetus auratus; RNA-Seq
library_construction_protocol: The right middle lobe of the lungs of infected animals was homogenized in 1 ml TRIzol Reagent (Ambion, Thermo Fisher Scientific) in Lysing Matrix M tubes (MP Bioscience, Hilton, UK) using the Precellys24 tissue homogenizer (bertin TECHNOLOGIES) for 2x 15 sec at 6,000 rpm. Samples were kept on ice all the time.  Organ debris was removed by subsequent centrifugation (13 min, 6,800 rpm, 4°C). Clear supernatant was used for RNA purification with Direct-zol RNA MiniPrep kit (Zymo research, Freiburg (Breisgau), Germany) according to the manufactures introduction. The isolated RNA samples were used for NNSR priming based RNA-Seq library preparation with the following modifications. Total RNA, was used for rRNA removal using the QIAseq FastSelect –rRNA HMR Kit (Qiagen) in combination with reverse transcription as follows. A 35 ul reaction mixture containing 1 ug RNA, 100 pmol NNSR_RT primer (gctcttccgatctctNNNNNN), 8 ul of 5x SuperScript IV buffer (Invitrogen) 20 pmol dNTPs and 1 ul of FastSelect-rRNA mix was subjected to the following hybridization protocol: 75°C 2 min, 70°C 2 min, 65°C 2 min, 60°C 2 min, 55°C 2 min, 37°C 5 min, 25°C 5 min, store at 4°C. For cDNA synthesis the reaction above was supplemented with dithiothreitol (10 mM), 20 units of RiboLock ribonuclease inhibitor (Thermo Fisher Scientific) and 200Us of SuperScript IV reverse transcriptase in a final reaction volume of 40 ul and incubated 45°C 5 min, 70°C 15 min.   The smears of 200-500 base pairs of the final barcoded libraries were purified from a 1.5% agarose gel and sequenced on a NextSeq 550 Illumina instrument using a single-end 86 bp setting. The RNS-Seq library preparation method used results in reads that start with the same two initial nucleotides. Hence, these were removed when performing quality- and minimum length- read trimming with the fastp algorithm with the default parameters.