GSM9364450: wild-type, involution, day 3; Mus musculus; RNA-Seq
Female C57BL6/J mice were bred with males to induce pregnancy. Right and left #4 mammary glands were isolated from 11-week-old (at time of harvest) females, either after 10 days post-parturition (Lac10), or after 10 days of lactation, followed by removing pups to force weaning and initiate involution for 3 days (Inv3). Inguinal lymph nodes were removed, and the mammary glands were digested to single-cell suspensions. Single Cells were resuspended in 1% UltraPure non-acetylated BSA (Invitrogen #AM2616) in Magnesium and Chlorine-free PBS, and stained with anti-EpCAM antibody (APC-Cy7, 1:200, BioLegend, #118218) or anti-CD45 antibody (FITC, 1:200, BioLegend #103107) for 30 minutes at 4°C. Cells were washed with 1% BSA-PBS and resuspended in 1% BSA-PBS containing a 1:1000 dilution of 7-AAD (Thermofisher Scientific: #A1310) for dead cell exclusion. UltraComp eBeads Plus Compensation Beads (Invitrogen, #01-3333-42), fluorescence minus one (FMO) stained cells, and unstained cells were also used as staining controls. Immediately, cells were run on a MoFlo XDP Flex cell sorter (Beckman Coulter) with a 100um nozzle, in conjunction with the CU Flow Cytometry Shared Resource. Debris, doublets, and dead cells were excluded, and FACS gates were set based on single stain, FMO, and unstained controls. Equal proportions of EpCAM+CD45- and CD45+ cells were sorted and pooled (70,000 of each cell type for a total of 140,000 cells) into 1% BSA-PBS buffer. Post-sort, cells were spun down (200rcf for 5 minutes) and resuspended in 0.1% BSA-PBS, then submitted to the UC Cancer Center (UCCC) Genomics Shared Resource for barcoding and library prep. Single-Cell 3' Gene Expression profile was obtained using the Chromium GEM-X Single-Cell 3' Library and Gel Bead Kit (10x Genomics). After cell counting and viability assessment, 20,000 viable flow-sorted cells were loaded on the Chromium X instrument (10x Genomics) for single-cell capture, followed by amplification of cDNA and library preparation to recover 15,000 cells per sample. Libraries were quantified, pooled, and sequenced on the NovaSeq X plus sequencer (Illumina) at 50,000 reads/cell at the University of Colorado Genomics Shared Resource.