title: GSM8759873: sample12, pigBAL, 135, DPI20, biol rep3; Sus scrofa; RNA-Seq
library_name: GSM8759873
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. Single cells were processed using the Chromium iX Controller with the Next GEM chip G and the single cell 3' kit v3.1 library preparation kit from 10x Genomics (Pleasanton, CA). For each of the BAL samples, approximately 10,000 cells were targeted for recovery during partitioning. The samples were grouped randomly and run in pairs, using three NextSeq High Output 150 cycle reagent kits (Illumina, San Diego, CA), with 1% PhiX, aiming for about 200 million reads per sample, with the run format aligning with 10X guidelines. The sequencing was performed on an Illumina NextSeq 550, using 3 high output 200 cycle reagent kit and flow cells.