GSM9442297: Sample3, Nulliparous, scRNAseq; Mus musculus; RNA-Seq
L2-5 and R2-5 mammary glands were harvested, minced, and chemically digested overnight in Advanced DMEM F/12 with 1% PSA, gentle collagenase/hyaluronidase, and DNAse I at 37C, 5% CO2, and added humidity. Partially digested glands were then mechanically digested by pipetting with a serological pipette until no tissue pieces were visible. Digested glands were washed with staining buffer (Hank's Balanced Salt Solution, 2% Bovine Calf Serum, 1% PSA) and centrifuged (1500RPM) at 4C for 5 minutes. Red blood cells were lysed with 5mL of ACK Lysis buffer for 5 minutes, and cells were washed with 15mL of staining buffer. Cells were treated with 0.25% Trypsin with EDTA and gently pipetted continuously for 2-3 minutes to digest the basement membrane. Cells were then treated with DNAse I and Dispase and pipetted continuously for 2-3 minutes to prevent clumping. The single-cell suspension was then filtered through a 40um mesh strainer and pelleted via centrifugation (1500RPM, 4C, 5 minutes). We performed scRNA-seq from mammary glands of 18m NP and 18m P mice using 10x chromium scRNA-seq (n=3 mice/group) as per manufacture's protocol using the 5' Gene expression workflow. Libraries were sequenced on the Illumina NovoSeq 6000 Platform by Medgenome, Inc.
GEO
GSM9442297_r1