title: GSM9161258: PyMT tumor total immune cells with vehicle treatment_2; Mus musculus; RNA-Seq
library_construction_protocol: Tumor tissues were prepared by mechanical disruption followed by 1-hour treatment with Collagenase Type 3 (280 U/ml, Worthington Biochemical,Cat. #LS004183) and DNase I (4 μg/ml,Merck#DN25) at 37 °C. After the digestion steps, the cell suspension was filtered through a 70 μM cell strainer. Cell surface staining was performed by incubating cells with specific antibodies for 30 mins on ice in the presence of CD16/CD32 mAb to block Fc binding. Live CD45+ immune cells were gated as DAPI-CD45+E-Cadherin-   by using BD FACS Aria III. The scRNA-seq libraries were constructed according to the protocol of the GEXSCOPER® Single-Cell RNA Library Kit (Singleron 4180011). Briefly, single cell suspension loaded onto the GEXSCOPE microchip which integrates single cell capture, cell lysis, capture of the cellular mRNA and molecular labeling. The barcoded cDNA was then amplified and used for the construction of single cell NGS libraries. The loading of the GEXSCOPE microchip was performed using the Singeron Matrix instrument. Individual libraries were diluted to 4 nM, pooled, and sequenced on Illumina NovaSeq 6000 with 150 bp paired end reads.