title: GSM7771943: WT mouse infected-1; Staphylococcus aureus; RNA-Seq
library_construction_protocol: RNA was isolated from punch biopsy homogenates of 1-day-old infection sites using the Direct-zol RNA extraction kit (Zymo), after treatment for 5 min with 50 μg/ml of lysostaphin. Isolated RNA was further treated with DNase according to the manufacturer's instructions (Ambion). A quantitative PCR (qPCR) assay was performed on the captured cDNA samples to determine the correct number of cycles required to generate the final sequencing library as well as to assess the efficacy of normalization and enrichment (73). Typically, higher threshold cycle (CT) values indicate greater depletion of host transcripts. The final library was created by PCR amplifying the samples with the full-length sequencing adaptors and 6-mer ScriptSeq (Epicentre) barcodes. Following PCR, the samples were cleaned and size selected in a two-step cleanup using 0.75 volumes of AMPure XP beads followed by 0.15 volumes to achieve a final library with an average size of ∼330 bp. Libraries were combined with 12 samples each, in equal amounts, and concentrated using DNA Clean & Concentrator-5 (Zymo Research). Prior to sequencing, the final libraries were quantified using qPCR. Combined libraries were sequenced on an Illumina NextSeq instrument using 75-cycle kits. Samples were loaded at 1.8 pM, and 75-base single-end reads were obtained.