GSM6208758: mammary glands, T=0 control; Mus musculus; RNA-Seq
Cre-injected glands were harvested 6, 12 or 18 weeks after injected. PBS-injected glands were harvested 12 weeks after injection. Glands/tumors were processed into a single cell suspension as described in the section 'flow cytometry analysis'. Samples were stained using the following antibodies: CD31- APC, CD45-AF700, EpCAM-FITC and CD49f-PE-Cy7. Fibroblasts were considered negative for all mentioned markers. Sorted fibroblasts were spun down and frozen in FCS + 10% DMSO until use. On day of scRNA sequencing, samples were thawed, checked for viability by flow cytometry (percentage of dapi-negative cells was above 75% for all samples). Cells were resuspended 1000 cells/ul in 1xPBS containing 0.04% weight/volume BSA (400 ug/ml) and for each sample the Chromium Controller platform of 10X Genomics was used for single cell partitioning and barcoding. Per sample each cell's transcriptome was barcoded during reverse transcription, pooled cDNA was amplified and Single Cell 3' Gene Expression libraries were prepared according to the manufacturer's protocol “Chromium Next GEM Single Cell 3' Reagent Kits v3.1” (CG000204, 10X Genomics). All four Single Cell 3' Gene Expression libraries were quantified on a 2100 Bioanalyzer Instrument following the manufacturer's protocol “Agilent DNA 7500 kit” (G2938-90024, Agilent Technologies). These Single Cell 3' Gene Expression libraries were combined to create one sequence library pool which was quantified by qPCR, according to manufacturer's protocol “KAPA Library Quantification Kit Illumina® Platforms” (KR0405, KAPA Biosystems). The NextSeq 550 Illumina sequencing system was used for paired end sequencing of the Single Cell 3' Gene Expression libraries at a sequencing depth of approximately 18.000 reads pairs/cell. NextSeq 550 paired end sequencing was performed using 28 cycles for Read 1, 8 cycles for Read i7 and 56 cycles for Read 2, using NextSeq 500/550 High Output Kit v2.5 (75 Cycles) Reagent Kit (PN 20024906, Illumina).