title: GSM7063583: WT_6A, scRNAseq; Mus musculus; RNA-Seq
library_construction_protocol: 4th mammary glands from WT or Gpm6aKO mice were isolated from 6-8 weeks old virgin female mice at specified stages. Minced tissue was placed in culture medium [RPMI 1640 with 25 mM HEPES, 5% FBS, 1% penicillin-streptomycin, 300 U/ml collagenase III (Worthington)] and digested for 2 h at 37◦C with shaking violently every 15 min. The cell pellet was collected by centrifugation at 1000 rpm, 4◦C for 5 min. After lysis of the red blood cells in NH4Cl, a single-cell suspension was obtained by sequential incubation with 0.05% trypsin-EDTA at 37◦C for 5 min and 0.1 mg/ml DNase I (Sigma) for 5 min with gentle pipetting, followed by filtration through 70-µm cell strainers. The total primary mammary epithelial cells (DAPI- cells) were FACS-isolated from three WT and three Gpm6aKO littermates. Cells were examined by microscope after 0.4% Trypan blue coloring. When the viability of cells was higher than 80 percent, the experiment of library construction was performed. The single-cell library was constructed using the ChromiumTM Controller and ChromiumTM Single Cell 3' Reagent Version 2 Kit (10x Genomics, Pleasanton, CA). Briefly, single cells, reagents and Gel Beads containing barcoded oligonucleotides were encapsulated into nanoliter-sized GEMs (Gel Bead in emulsion) using the GemCode Technology. Lysis and barcoded reverse transcription of polyadenylated mRNA from single cells were performed inside each GEM. Post RT-GEMs were cleaned up and cDNA were amplified. cDNA was fragmented and fragments end were repaired, as well A-tailing was added to the 3' end. The adaptors were ligated to fragments which were double sided SPRI selected. Another double sided SPRI selecting was carried out after sample index PCR. The final library was quality and quantitated in two methods: check the distribution of the fragments size using the Agilent 2100 bioanalyzer, and quantify the library using real-time quantitative PCR (QPCR) (TaqMan Probe).