title: GSM6745547: YUMMER1.7 tumor; Mus musculus; RNA-Seq
library_construction_protocol: Cell suspensions from YUMMER1.7 tumors (n=3) were created by mincing tumors followed by enzymatic digestion with 10 mg/ml collagenase D and 80 U/ml DNase for 20 minutes at 37C with shaking. Suspensions were then passed through a 70um cell strainer and washed with PBS. Cell suspensions were  immunostained with CD45-APC antibodies and then CD45+ cells enriched for Miltenyi APC microbeads and the QuadroMACs Magnetic Seperator and LS Columns. Enriched CD45+ cell suspensions were stained with DAPI and then sorted on SY3200 HAPS sorter to improve purity of live CD45+ cells. 2.5e5 cells were sorted from each tumor and then pooled. Library prep was performed according to 10x Genomics single cell 3' kit (v3.1) protocol. Briefly, GCs were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.