GSM7792115: LN CD45- cells, mock-1; Mus musculus; RNA-Seq
At 8 h post-infection the draining popliteal lymph node from mock- or CHIKV-inoculated mice was pooled into individual replicates (2 replicates; LNs from 5 mice pooled per replicate). Lymph nodes were mechanically homogenized using a 22G needle in Click's medium (Irvine Scientific, 9195) supplemented with 5 mg/mL liberase DL (Roche, 05401160001) and 2.5 mg/mL DNase (Roche 10104159001) for 1 h at 37°C. After incubation, digested LNs were clarified by passing through a 100-μm cell strainer. Cell suspensions were enriched for CD45- cells by labeling cells with PE-conjugated anti-mouse CD45 (30-F11), CD140A (APA5), and Ter119 (Ter119) monoclonal antibodies and subsequent depletion of PE-labeled cells using Miltenyi anti-PE microbeads (130-048-801) and Miltenyi MACS LS (130-042- 401) columns according to the manufacturer's instructions with the following modifications: (i) We used 25% of the recommended volume of anti-PE microbeads, and (ii) we subjected the CD45- enriched cell fraction to a second MACS LS column. All cell suspensions post-column enrichment were enumerated using a hemacytometer. Cell fractions throughout the procedure were analyzed for PE-labeled cell depletion and enrichment of CD45- cells by flow cytometry. Next GEM single-cell 3′ GEM library and gel bead kit v3.1 (1000128)