GSM6284585: MMC, replicate 1, scRNAseq; Mus musculus; RNA-Seq
Embryonic day 18.5 old fetuses were isolated from the maternal uterus and sacrificed by decapitation. Fetal brains were then disrupted mechanically and washed through a 40µm cell strainer with PBS. Unspecific FcγRII/III binding was blocked by incubating cells with normal rat serum and TruStain fcX (anti-mouse CD16/32, BioLegend, Cat. No. 101320). Afterwards, cells were stained with Fixable viability dye eFluor506TM, anti-CD45.1 (1:400, BioLegend, Cat. No. 110705), anti-CD45.2 (1:100, BioLegend, Cat. No. 109823), anti-H-2Db/b (1:20, BioLegend, Cat. No. 111507), anti-H-2Dd/d (1:200, BioLegend, Cat. No. 114712). Cells were sorted using the FACSAria III flow sorter (BD Life Sciences) with the Diva Software. Single cells were encapsulated in droplets using GemCode Technology and processed by using Chromium Single Cell 5' Reagent Kits (v2, 10X Genomics) following manufacturer's specifications. Briefly, transcriptome from each cell was captured in single droplet, and hybridized to gel beads with unique cell barcode and unique molecular identifier (UMI). Subsequently, barcoded cDNA was transcribed in droplets and then released for library construction. The constructed libraries were sequenced by HiSeq 4000 (Illumina) in paired-end to reach approximately 50.000 reads per cell.