GSM5583808: Mixed chimera CD45.2 myeloid fraction scRNAseq; Mus musculus; RNA-Seq
Lungs were perfused and processed into a since cell suspension which was immunostained with a cocktail of fluorescent antibodies for FACS-sorting. For each scRNA-seq experiment, we sorted around 100000 total myeloid CD45.1+ or CD45.2+ cells as further defined as described in the text. Single cell suspensions were pelleted and resuspended in 100 ul diluted hashtag antibodies at 4˚C for 20 minutes (Hashtag antibody conjugated as per New York Genome Center hashing protocol https://citeseq.files.wordpress.com/2019/02/cell_hashing_protocol_190213.pdf ). Stained cells were washed three times in PBS + 0.5% BSA by 4˚C centrifugation at 350 RCF to remove unbound antibodies. Washed cells were resuspended in 150ul wash buffer and counted using a Nexcelom Cellometer Auto2000. Hashed samples were pooled in equal amounts of live cells.   Volume of cell suspension was adjusted to achieve a target of 2 million cells/mL. Hashed sample pool was counted and loaded on two lanes of the 10x Genomics NextGem 5'v1.1 assay as per the manufacturer's protocol with a targeted cell recovery of 20,000 cells. Gene expression libraries were made as per the 10x Genomics demonstrated protocol. During cDNA amplification, hashtag oligonucleotides (HTO) were enriched during cDNA amplification.