GSM6911960: mixed sample of mammary gland - WT, K14-cKO, MMTV-cKO; Mus musculus; RNA-Seq
The fresh mammary gland tissues were washed with Hanks Balanced Salt Solution (HBSS) for 3 times and minced into 1-2 mm pieces. Then the tissue pieces were digested with 2ml GEXSCOPE® Tissue Dissociation Solution (Singleron) at 37℃ for 15min in 15ml centrifuge tube with sustained agitation. After digestion, using 40-micron sterile strainers to filter the samples and centrifuging the samples at 1,000 rpm for 5 minutes. Then the supernatant was discarded, and the sediment was resuspended in 1ml PBS (HyClone). To remove the red blood cells, 2 mL GEXSCOPE® red blood cell lysis buﬀer (Singleron) was added at 25°C for 10 minutes. The solution was then centrifuged at 500 × g for 5 min and suspended in PBS. Cells were incubated with 7-AAD (1:1000, Invitrogen) on ice for 30 minutes, and sorted by flow cytometry to filter dead cells. Single-cell suspensions with 1×105 cells/mL in concentration in PBS (HyClone) were prepared. Single-cell suspensions were then loaded onto microfluidic devices and scRNA-seq libraries were constructed according to Singleron CLindex® protocol by GEXSCOPE® Single-Cell RNA Library Kit (Singleron Biotechnologies). mixture libraries were diluted to 4nM and pooled for sequencing. Pools were sequenced on Illumina HiSeq X with 150 bp paired end reads.