title: scDNA-seq of UMM069: Sample S4
design: "Immediately following surgical eye removal or liver resection, the tissue was dissected to isolate the tumour region for single cell dissociation.  Metastatic tumour tissue was intentionally sampled far from the tumour-liver interface to avoid contaminating normal liver tissue. Tissue samples were placed immediately in gentleMACS C tubes (Miltenyi Biotec) containing 5 mL of DMEM or RPMI1640 Media with 10% FBS and 400U/mL of collagenase IV for digestion. The ÒDissociation of soft tumorsÓ protocol from the Miltenyi Tumor Dissociation Kit was used with a slight modification. Briefly, samples were processed using a gentleMACS dissociator (Miltenyi Biotec) using program Òh_tumor_01Ó and incubating at 37C for 1 hour. Samples were processed again using program Òh_tumor_03Ó and passed through a 70 _m cell strainer (Miltenyi Biotec). After the initial incubation step cells were kept on ice for the remainder of the protocol. Wide Orifice 1 mL Pipet Tips (VWR) were used to prevent cell shearing. The cell suspension then underwent a Debris Removal Solution (Milteni Biotec) protocol, a density gradient method to remove dead cells and debris. The samples were resuspended in D-PBS containing 0.1% BSA and filtered into Falcon Tubes (12 x 75 mm) with Cell Strainer Cap (BD). An aliquot of the singe cell suspension was stained with the LIVE/DEADª Viability/Cytotoxicity Kit for mammalian cells (Invitrogen) to ensure viability was greater than 85%. Single-cell DNA sequencing was performed using the Chromium instrument. Single cell suspensions were counted using both the Cellometer K2 Fluorescent Viability Cell Counter and a haemocytometer and adjusted to 1,000 cells/µl. Samples were run using the Chromium Single Cell DNA Library & Gel Bead Kit (10X Genomics) with a target capture of 500 cells. The samples were processed on Chromium Single Cell C and D Chips (10X Genomics) according to the manufacturerÕs protocol and subsequently run on a thermocycler. Single-cell genomic DNA libraries were sequenced using the NextSeq 550."
attributes.tag: alignment_software
attributes.value: Cellranger DNA 1.0.0