GSM4980336: Breast Tumor Platform 60 min; Mus musculus; RNA-Seq
Kidneys and mammary tumor were dissected, minced into ~ 1 mm3  pieces, and processed using either the microfluidic platform (15 and 60 min digestion device intervals, single pass through integrated dissociation/filter device) or control (60 min digest) using 0.25% type I collagenase. Recovered cells were centrifuged (400xg, 5 min), treated with 100 Units of DNase I for 5 min at 37°C, and washed by centrifugation into PBS+. Samples were then incubated with RBC lysis buffer for 5 min on ice, centrifuged, and resuspended in PBS+. Cells were stained with SytoxBlue (Life Technologies, Carlsbad, CA, USA) prior to FACS (FACSAria Fusion, BD Biosciences, Franklin Lakes, NJ) to remove dead cells and ambient RNA. Sorted live single cells (SytoxBlue-neg) were centrifuged and resuspended at a concentration of 1000 cells/µL in PBS with 0.04% BSA. The 10x Chromium system (10x Genomics, Pleasanton, CA) was then used for droplet-enabled scRNA-seq. Oil, cells, reagents, and beads were loaded onto an eight-channel microfluidic chip. Lanes were loaded with ~17,000 cells from each of the samples, determined using an automated cell counter (Countess II, Invitrogen, Carlsbad, CA). Library generation for 10x Genomics Single Cell Expression v3 chemistry was performed according to manufacturer's instructions.