Instrument: NextSeq 500
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: PAIRED
Construction protocol: Spleens were homogenized in 1 mL TRIzol reagent using TissueRuptor (Qiagen) and total RNA was extracted following TRIzol method (Invitrogen). RNA concentrations were quantified using a NanoDrop spectrophotometer (NanoDrop). Approximately 10 mg total RNA for each of the 12 rat samples was treated with TurboDNase (ThermoFisher Scientific) and ribosomal RNA (rRNA) depletion was performed using a Ribo-ZeroTM Gold rRNA removal kit (Illumina). For RNA-seq, we prepared strand-specific libraries by following the protocol from Zhang and colleagues (Zhang, Theurkauf et al. 2012). The quality of the prepared libraries was confirmed using the Advanced Analytical Technologies, Inc. Fragment Analyzer through the Molecular Biology Core Lab (UMass Medical School). The 12 libraries were pooled and sequenced with paired end reads (75 bp each) using Illumina NextSeq 500 according to the manufacturer's specifications.