title: GSM8171459: PlusMinus; Mus musculus; RNA-Seq
library_construction_protocol: After removal of EDTA fraction, samples from 5 Vil-Cre (Heterozygotes,HE), Rosa26R-Tomato (HE) mice were treated with collagenase (0.132mg/ml) Dispase (0.66mg/ml) and DNase1 (0.05mg/ml) solution for 35 min at 37°C, washed 2 times with BCM-2mM EDTA solution with centrifugation at 500g for 2min in-between. The cell pellet was washed once with PBS-2%FBS (FACS solution) and centrifuged at 500g for 2min. The cells were then incubated with rat anti-mouse CD16/CD32 (blocking solution) at a final dilution of 1/50 (v:v) in FACS solution for 15 min. The APC rat anti-mouse CD31 (1/200), APC rat anti-mouse CD45 (1/500) and BV711 rat anti-mouse CD326 (Epcam,1/200) antibodies were added to blocking solution and incubated for 45 min on ice. After 3 washes with FACS solution, DAPI was added to mark the dead cells (1/2000) and the cells were sorted using the FACS Aria I cytometer (BD Biosciences). Twenty thousand Epcam-positive and Tomato-negative (PlusMinus) or Epcam-positive and Tomato-positive (PlusPlus) cells were collected for each animal. These cells were processed through the Chromium Next GEM Single Cell 3' Reagent Kits v3.1 (10X Genomics) according to manufacturer's recommendations and sequenced on Novaseq6000 (Illumina). Library was performed according to the manufacter's instructions (single cell 3' v2 protocol, 10x Genomics).