GSM7295272: MFP, replicate 3; Mus musculus; RNA-Seq
Fresh tumor tissue was collected from sacrificed mice following microdissection, and all the subsequent steps were performed on ice, if not stated otherwise. Tumor tissue was placed in serum-free media and chopped into small pieces with scissors. The Tumor Dissociation Kit for mouse (Miltenyi, Cat# 130-096-730) was used to generate a single cell suspension. After the tissue digestion, cells were filtered through a 40 μm mesh filter and washed. Samples were stained with mouse Fc block and afterwards with anti-CD11b (M1/70) and anti-CD45 (30-F11). Dead cells were excluded using DAPI (Thermo Fisher Scientific, Cat# 62247) and viable cells were additionally stained with RedDot™1 Far-Red Nuclear Stain (Chemie Brunschwig Cat# BIO40060-1) following the manufacturer's instruction manual. Total immune cells were sorted as CD45+ DAPI- RedDot-1+ cells using an Aria II (SORP) instrument (BD) into 1.5 ml tubes containing 8 μl cold HBSS. Sorted cells were counted and a total of 16,000 total cells were loaded onto the 10x Chromium Controller following the manufacturer's instruction manual. For the generation of Gel Beads in Emulsion (GEM), Chromium Single Cell 3ʹ GEM Kit v3 (10x, 1000077) and the Chromium Single Cell B Chip Kit (10x, 1000074) were used. Gene expression libraries were prepared using the Chromium Single Cell 3ʹ Library Kit v3 (10x 1000078) following the manufacturer's instruction manual. The quantity of constructed libraries was determined with the Qubit Fluorometric Quantification (Thermo Fisher Scientific, Q32851). Library quality was determined using High Sensitivity Next-Generation-Sequencing Fragment Analysis (Agilent Technologies, DNF-474-0500) and the sequencing was performed by Genewiz Services (http://www.genewiz.com/) on an Illumina HiSeq 2500 or Illumina NovaSeq6000 S4 Flow Cell using a 10x sequencing configuration. Targeted sequencing depth was > 20,000 reads per cell.