title: GSM4830544: T4 Mammary_gland_tumour; Mus musculus; RNA-Seq
library_construction_protocol: Tumours where collected from Young adult (13-14-week-old) female MMTV-PyMT::K14Cre::Rosa-tdTomato mice, minced manually using sterile scalpels and further finely cut until homogeneous aspect using McIlwain Tissue Chopper (TED PELLA, INC). Minced samples where digested in digestion buffer (2,5ml for 1mg tumour) at 37ºC for 45 minutes on rocking plates chamber (reference VWR). Digestion buffer was based on PBS supplemented 2.5 Wünsch unit's TH Liberase /ml (Roche) (50mg TH Liberase in 52 PBS = 5W unit/ml) and with 25μg/ml DNase I (Roche). To achieve single cells disruption, tumours fragments were pipetted up and down every 5 minutes. The progression of the cellular disaggregation was evaluate using microscope. Disaggregated samples were neutralized with 15ml breast medium supplemented by 25μg/ml DNase I and passed through a 70μm filter (BD Falcon) then 40μm filter (BD Falcon). Further, cells were spun down (5 minutes at 400 RCF at RT) and the pellet resuspended in 5ml of red blood lysis ACK (Ammonium-Chloride-Potassium) buffer for 5 minutes at RT with continuous gentINle rotation. After, cells were spun down and resuspended in 5ml FACS buffer/5mM EDTA and filtered through 40μm filter. 20μl of the cell preparation where mixed with equal volume of Trypan Blue and incubated for 5min to evaluate cells number and confirm the quality of single cells preparation, cell viability and low debris contains. Cells were further spun down (3 minutes at 400 RCF at RT) and resuspended in Dead cell removal beads (MiltenyiBiotec) at 100μl/10M and Incubated 15min at RT with gentle mixing. During the incubation, MACS LS column (MiltenyiBiotec) were placed on the magnet rack and conditioned by rinsing 1X with binding buffer 1X. After, cells were spun down (3 minutes at 400 RCF at 4C), resuspended in 3ml 1X binding buffer and applied on the magnetic column. Passing living cells (column retain the dead cells) were collected in 50ml Falcon tube placed in ice. Column where rinsed 4X with 3ml binding buffer to further living cells in the same Falcon tube. Collected cell were finally spun down (5 minutes at 400 RCF at 4C) and resuspended in cold breast medium and kept on ice. 20μl cells where mixed with 20ul trypan Bleu, incubate for 5minutes to count the proportion of viable and double check single cells state. Viability was first time evaluated on a BD FACS Aria III % as well as cancer cells, non-cancer cells and debris contains, and later, by measuring DAPI negative cells fraction. Finally, cells concentration was adjusted to 900-1000 cell/μl with cold FACS buffer and directly used for GEM (Gel Bead-In-Emulsions) preparation. Individual cell encapsulation for single cell expression profiling was done using 10x Chromium Controller (10xGenomics). For every sample, single cell suspension was loaded into a Chromium Single Cell A Chip (10x Genomics) and processed following the manufacturer's instructions. Single-cell RNA-seq libraries were prepared using the Chromium Single Cell 3' Library & Gel Bead kit v2 and samples were multiplexed using Chromium i7 Multiplex kit (10x Genomics). Pooled libraries were then loaded on a HiSeq2500 instrument (Illumina) and sequenced to obtain 75 bp paired-end reads.