Macs negative selection kit

Gene expression profiling was performed as shown previously (Barnitz et al., 2013 (link); Penaloza-MacMaster et al., 2015 (link); Quigley et al., 2010 (link); Wang et al., 2019 (link)). In brief, C57BL/6 mice were intramuscularly immunized with 10⁴ PFU of LCMV, and at day 7, splenic CD8 T cells were MACS (magnetic activated cell sorting) sorted with a MACS negative selection kit (STEMCELL). Purified CD8 T cells were stained with D^bGP33 tetramer, live dead stain, and flow cytometry antibodies for CD8 and CD44 to gate on activated CD8 T cells. Live, CD8⁺, CD44⁺, and D^bGP33⁺ cells were FACS sorted to ∼97% purity on a FACS Aria cytometer (BD Biosciences) and stored at -80°C in 1 ml of TRIzol (Life Sciences). RNA extraction was performed with the RNAdvance Tissue Isolation kit (Agencourt). RNA quality and RNA-Seq downstream analyses were performed at the NUSeq core at Northwestern University. For analysis, adapters were trimmed from reads using cutadapt version 1.13 and aligned to 10 mm using STAR version 020201. For gene counting, htseq-count version 0.6.1p1 was used, and differential expression analysis was conducted using DESeq2 version 1.14.1. RNA-Seq data were uploaded into the GEO database (accession no. GSE129827) in a record titled “Gene expression comparison of splenic virus-specific CD8 T cells after infection with LCMV vector and treatment with IgG or aIFNAR1.”