GSM6879661: Sorted Kaede-green myeloid cells, isotype control treated tumours; Mus musculus; RNA-Seq
Tumours were cut into small pieces using surgical scissors then digested using Miltenyi Biotec Tumour Dissociation Kit using a gentleMACS Dissociator for 40 minutes at 37C, as per manufacturer's protocol. Subsequently, the sample was filtered through a 70 μm strainer to remove undigested tissue debris. Next, using Miltenyi Biotech Dead Cell Removal Kit and LS Columns, as per manufacturer's instructions, dead cells were removed from samples. Lymph nodes were cleaned and dissected in RPMI 1640 medium and crushed through a 70 μm strainer. Thereafter, cells were stained for surface markers and LIVE/DEAD viability dye before being centrifuged at 400 g at 4°C for 5 min and resuspended in FACS staining buffer for sorting (BD FACS Aria II Cell Sorter). Gene expression libraries from were prepared from FACS-sorted populations of single cells using the Chromium Controller and Single Cell 3' Reagent Kits v3 (10x genomics, Inc.) according to the manufacturer's protocol. The resulting sequencing libraries comprised of standard Illumina paired-end constructs flanked with P5 and P7 sequences. The 16 bp 10x barcode and 10 bp UMI were encoded in read 1, while read 2 was used to sequence the cDNA fragment. Sample index sequences were incorporated as the i7 index read. scRNA-seq, 10x Genomics Chromium GEM 3' v3; 2 x 150bp paired-end sequencing performed on NovaSeq 6000 (Illumina)