RNA-Seq of Mus musculus: spleen from adult female mice at 4 weeks post inactivated VarV immunization
Total RNA was extracted following the Genewizs  (Suzhou, China) protocol. RNA purity, concentration, and integrity were assessed. 1 g of total RNA per sample was used for library preparation. Poly(A) mRNA was isolated using Oligo(dT) beads and fragmented with divalent cations under high temperature. First-strand and second-strand cDNA synthesis was performed using random primers. The resulting double-stranded cDNA was end-repaired, A-tailed, and ligated with sequencing adaptors. Size selection was performed using DNA Clean Beads. Libraries were amplified by PCR with P5 and P7 primers, and products were quality-checked prior to sequencing. Indexed libraries were pooled and sequenced on an Illumina HiSeq, NovaSeq, or MGI2000 platform using a 2  150 bp paired-end configuration, according to the manufacturers instructions.