title: GSM5583658: Trem2 Knockout scRNAseq; Mus musculus; RNA-Seq
library_construction_protocol: Lungs were perfused and processed into a since cell suspension which was immunostained with a cocktail of fluorescent antibodies. For each scRNA-seq experiment, we sorted around 100000 total myeloid and/or lymphoid 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 with the addition of 3 pmol of a HTO Additive primer (5'GTGACTGGAGTTCAGACGTGTGCTC).