GSM7232745: lactating mammary gland p7 1; Mus musculus; RNA-Seq
Mice were sacrificed by CO2 inhalation and intracardially perfused with phosphate buffered saline. After perfusion, left and right abdominal mammary glands were dissected, and inguinal lymph nodes were removed. All the samples were cut into small pieces in an Eppendorf tube, followed by digestion in 0.4 mg/ml collagenase type IV and 0.04 mg/ml DNase I in Ca2+/Mg2+ HBSS supplemented with 10% FCS for 40 mins at 37°C, while shaking. Samples were homogenized with a 18G needle and a syringe and filtered through a 100 µm cell strainer to obtain a homogeneous cell suspension. Cells were once washed in PBS, resuspended in red blood cell lysis and incubated on ice for 5 min, then filtered through 100 μm cell strainer and washed with PBS. Then cells were first incubated with a-CD16/32 antibody to block FcR receptors, and stained with an antibody mix as specified in the "Methods" section of the paper. Following staining, populations of interest were sorted on BD FACS Aria III or BD S6 equipped with a 100 µm nozzle (the gating strategies are specified in the paper). The sorted populations were then washed, resuspended in PBS and ready for scRNAseq sample preparation steps. Human milk samples were collected at the University Hospital Zurich from healthy women, aged 20 to 40 years [mean (SD) 32.0 (5.0)], within 4 to 63 days after delivery. For isolation of milk cells, milk was diluted in a 1:1 ratio with PBS and centrifuged at 800 g for 20 minutes at 15°C. The lipid layer and skim milk were removed, and the cell pellet was washed twice in PBS by centrifugation at 400 g for 5 minutes and resuspension in PBS. for p8p11p14 samples, libraries were constructed using 10X 3' scRNA-seq v3.1 with Hashtags with dual-indexing; for the p7 samples, libraries were prepared using the 10X Genomcis GEX 3`v3.1 Single index kit