GSM5617484: D9muD10mu VACV 18 hr rep 1; Homo sapiens; RNA-Seq
Total RNA was extracted from samples using TRIzol and treated with TURBO DNase. Afterwards, the RNA was isolated using RNA Clean & Concentrator - 5 kit, and 2μL of ERCC RNA spike-ins (1:100 diluted) was added to each sample. RNA-Seq libraries were prepared using services provided by QB3 UC Berkeley. Illumina Ribo-Zero rRNA Removal Kits were used to deplete the RNA. Subsequent   library preparation steps of fragmentation, adapter ligation and cDNA synthesis were  done on the depleted RNA using the KAPA RNA HyperPrep kit (KK8540).   Truncated universal stub adapters were used for ligation, and indexed primers were  used during PCR amplification to complete the adapters and to enrich the libraries for  adapter-ligated fragments. Samples were checked for quality on an Agilent  Fragment Analyzer. Samples were then transferred to the Vincent J. Coates Genomics  Sequencing Laboratory (GSL), another QB3-Berkeley Core Research Facility at UC Berkeley,  where Illumina sequencing library molarity was measured with quantitative PCR with the Kapa  Biosystems Illumina Quant qPCR Kits on a BioRad CFX Connect thermal cycler. Libraries were then  pooled evenly by molarity and sequenced.