title: TCR enrichment of scRNA-seq of UMM066: Sample S21
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 RNA sequencing was performed using the Chromium (10X Genomics) instrument. Single cell suspensions were counted using both the Cellometer K2 Fluorescent Viability Cell Counter (Nexcelom) and a haemocytometer and adjusted to 1,000 cells/µl.  The manufacturerÕs protocol was used with a target capture of 10,000 cells. Each sample was processed on an independent Chromium Single Cell A Chip (10X Genomics) and subsequently run on a thermocycler (Eppendorf). B- and T- cell VDJ libraries were sequenced on a MiSeq instrument."
attributes.tag: alignment_software
attributes.value: Cellranger 2.2.0