GSM4476801: Mock-infected lung, 1; Homo sapiens; RNA-Seq
Organoid infections were performed at an MOI of 0.1 and harvested at 24 hpi in DMEM supplemented with 0.3% BSA, 4.5 g/L D-glucose, 4 mM L-glutamine and 1 μg/ml TPCKtrypsin. Total RNA was extracted in TRIzol (Invitrogen) and DNase I treated using Directzol RNA Miniprep kit (Zymo Research) according to the manufacturer's instructions.  cDNA libraries were sequenced using an Illumina NextSeq 500 platform. The resulting single end reads were checked for quality (FastQC v0.11.5) and processed using the Digital Expression Explorer 2 (DEE2) (Ziemann et al., 2019) workflow.  Adapter trimming was performed with Skewer (v0.2.2) (Jiang et al., 2014).  Further quality control done with Minion, part of the Kraken package (Davis et al., 2013).  The resultant filtered reads were mapped to human reference genome GRCh38 using STAR aligner (Dobin et al., 2013) and gene-wise expression counts generated using the “-quantMode GeneCounts” parameter.  BigWig files were generated using the bamCoverage function in deepTools2 (v.3.3.0) (Ramirez et al., 2016). RNAseq libraries of polyadenylated RNA were prepared using the TruSeq RNA Library Prep Kit v2 (Illumina) or TruSeq Stranded mRNA Library Prep Kit (Illumina) according to the manufacturer's instructions.