title: GSM4511884: active_KR71; Macaca mulatta; RNA-Seq
library_construction_protocol: At necropsy lung tissues were collected and digested with DNase I and Liberase at 37ºC for 30-45 mins. Digested lung tissue was gently dispersed by passage through a 70 μm pore size nylon tissue strainer (Falcon; BD Biosciences); the resultant single-cell suspension was treated with ACK lysis buffer to remove any residual RBC, washed twice, and counted, as previously described (Foreman et al., 2016; Gautam et al., 2018; Kaushal et al., 2015; Kuroda et al., 2018; Mehra et al., 2015). Cells prepared in this way were stored at -70ºC and then used for downstream processing of scRNA-seq. scRNA-seq was done according to the manufacturer instructions (10X genomics). Briefly, after quickly thawing the frozen lung single cell suspension in water bath, 2X106 cells were taken for downstream processing. Cell suspensions were enriched for CD45 by using NHP CD45-microbeads according to the manufacturer instruction (Milteny Biotec). After then, CD45+ enriched lung single cell suspensions were subjected to droplet-based massively parallel single-cell RNA sequencing using Chromium Single Cell 3' (v3) Reagent Kit in the BSL-3 level laboratory as per manufacturer's instructions (10x Genomics). Briefly, cell suspensions were loaded at 1,000 cells/μL with the aim to capture 10,000 cells/lane. The 10x Chromium Controller generated GEM droplets, where each cell was labeled with a specific barcode, and each transcript labeled with a unique molecular identifier (UMI) during reverse transcription. The barcoded cDNA was isolated and removed from the BSL-3 space for library generation. The cDNA underwent 11 cycles of amplification, followed by fragmentation, end repair, A-tailing, adapter ligation, and sample index PCR as per the manufacturer's instructions. Libraries were sequenced on a NovaSeq S4 (200 cycle) flow cell, targeting 50,000 read pairs/cell.
attributes.tag: GEO Accession
attributes.value: GSM4511884