title: GSM7966152: sample1, pigBAL, PBS, rep1; Sus scrofa; RNA-Seq
library_construction_protocol: Cryopreserved BAL cells were thawed, washed and resuspended in PBS. Cells were incubated with antibodies against CD4 (clone 74-12-4, PerCP-Cy5.5-conjugated, BD Biosciences), CD8β (clone PPT23, FITC-conjugated, Bio-Rad), CD3 (clone PPT3, APC-conjugated, Southern Biotech), CD16 (clone G7, AF647-conjugated, Bio-Rad), CD172a (clone 74-22-15, PE-conjugated, Bio-Rad) and Fixable Viability Dye eFluor780 (ThermoFisher). After two washes in PBS, cells were fixed, washed again and sorted on a FACSAria UIII (BD Biosciences), equipped with 405, 488, 561 and 640nm lasers and BD FACSDiva 8 software. After gating on FSC-AdimSSC-Adim cells and exclusion of doublets and dead cells, CD4+, CD8β+ and CD3-CD16- (to enrich for B cells) cells were sorted (12,000 cells per population). In addition, after gating on FSC-AbrightSSC-Abright cells, doublet and dead cell exclusion and gating on CD16+CD172a+ cells, 12,000 macrophages were sorted. Cells from each fraction were pooled and subjected to partitioning and barcoding on a 10x Genomics Chromium iX Controller. For 10X, single cells were processed with the Chromium iX controller, using the Next GEM chip G, and single cell 3' kit v3.1 library preparation kit (10X Genomics, Pleasanton, CA). An estimated 10,000 cells were targeted for recovery during partitioning. Samples were randomly grouped and run with two libraries per sequencing run on three NextSeq High Output 150 cycle reagent kits (Illumina, San Diego, CA), with 1% PhiX, targeting approximately 200 million reads per sample.