GSM7822082: S4, biol rep 1, Treg, scRNAseq; Mus musculus; RNA-Seq
Uterine tissue was minced and suspended in digest solution (HBSS + 10% FBS + 1mg/ml Collagenase IV) in a 37°C water bath for 30 minutes. Digested tissue was passed through 40mm cell strainers. Red blood cells were removed with ACK lysing buffer and incubation at room temberature for 10 minutes, rinsed with HBSS. CD11b+ cells were enriched using CD11b+ microbeads follwoing the manufacturer's protocol (Miltenyi Biotec). Cells were stained with Live/Dead Fixable Aqua for 30 minutes. Cells were then stained with the following extracellular antibody mix, all diluted 1:400 in FACS buffer (PBS + 2% FBS + 20mM EDTA):  anti-CD45-SB702 (eBioscience); anti-CD11b-PE, anti-CD3e-APC-Cy7, and anti-CD19-APC.Cy7 (BioLegend) for 30 minutes. Cells were sorted on a BD FACS Aria, collecting live, singlet, CD45+, CD11b+ cells in 50% FBS + 50% PBS. Cells were resuspending in 1000 cells/1ml sterile PBS for library preparation. 10X Genomics Single Cell 3' version3 was used and manufacturer's protocol was followed. Briefly, cDNA was created from uterine myeloid cells using Chromium single cell 3' v3 gel beads and RT mix, which use a primer containing the illumina R1 primer, UMI, 10X barcode and poly(dT) sequence.  Dynabeads MyOne Silane were used to purify cDNA from GEM/RT mix. cDNA was amplified by PCR followed by SPRI enzyme fragmentation for ideal sizing. P5, P7, a sample index and R2 primers were then added via adaptor ligation to create a gene expression library with paired-end constructs begining and ending with P5 and P7.