GSM6212293: p21094-s025_RRc17-PBMC-Day-7-2021-03-23; Macaca mulatta; RNA-Seq
GSM6212293
PBMCs: Whole blood was collected from the femoral vein in EDTA tubes. Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood using a Ficoll-Paque Premium density gradient and resuspended in R10 media. For bulk RNAseq, 250,000 PBMCs were frozen in 700uL of QIAzol.; BAL: To collect BAL, a fiberoptic bronchoscope was used to administer 0.9% NaCl solution into the bronchus and re-aspirate the BAL fluid. BAL was then filtered through a 70μm cell strainer and centrifuged. Following centrifugation, BAL fluid supernatant was removed and the remaining cell pellet was lysed in ACK lysing buffer and resuspended in R10. For bulk RNAseq, 50,000 BAL cells were frozen in 700uL of QIAzol. For 10X, ~125,000 BAL cells from each animal were incubated with one of five TotalSeq-C anti human Hashtags. Following a 30 minute incubation, cells were washed with PBS, filtered with a 40uM cell filter, and resuspended in R10. Ten nanograms of total RNA was used as input for cDNA synthesis using the Clontech SMART-Seq v4 Ultra Low Input RNA kit (Takara Bio). Amplified cDNA was fragmented and appended with dual-indexed bar codes using the NexteraXT DNA Library Preparation kit (Illumina).