GSM8898372: GC Day 4; Macaca mulatta; RNA-Seq
Cells were resuspended in 100uL of live dead NIR 780 (Thermo Cat#L34994), diluted 1:1000 in PBS and incubated for 15 minutes. Cells were washed and stained with 50uL surface stain antibodies diluted in PBS + 1% FBS and incubated for 20 min at 4°C. Cells were washed 3 times with PBS + 1% FBS, before fixation with eBioscience Intracellular Fixation & Permeabilization Buffer Set (Thermo Cat# 88-8824-00) for 16 hours at 4°C. After fixation cells were washed with eBioscience Permeabilization Buffer, resuspended in 50uL intracellular stains diluted in Perm. Buffer, and stained for 30 min at 4°C. After staining cells were washed with Perm. Buffer 2x and resuspended in 180uL of PBS + 1% FBS + 0.05% Sodium Azide for analysis on the Cytek Aurora 5L. Cryopreserved BAL cells were processed for scRNAseq using the 10X Genomics Chromium Single Cell 3′ kit (v3.1). Samples were then stained with unique TotalSeq-A hashtag antibodies (HTO) as per manufacturer's (Biolegend) protocol. Equal number of cells from each sample were pooled and super-loaded on a 10X Genomics Next GEM chip and single cell GEMs were generated on a 10X Chromium Controller. Subsequent steps to generate cDNA and HTO libraries were performed following 10X Genomics and Biolegend's protocol respectively. Libraries were pooled and sequenced on an Illumina NovaSeq. Sequenced data was processed using cellranger (version 7.1.0) to demultiplex the libraries. The reads were aligned to mmul_10 genome to generate count tables. Sample batch effects were corrected using Harmony R package (version 1.2.3). The count tables were then further processed and analyzed using the Seurat (version 5.2.1) in R (version 4.4.1).