Instrument: Illumina HiSeq 2500
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: PAIRED
Construction protocol: Animals at endpoint were euthanized by asphyxiation with CO2 followed by cervical dislocation and perfusion with 10mM EDTA in D-PBS. Prior to perfusion, Evan's Blue (Sigma-Aldrich, Cat. No. E2129-10G) was injected into the footpads and ears of anesthetized mice to aid in lymph node visualization. Solid tissues from the mice, which includes the primary tumour, lung, and lymph nodes were processed for flow cytometry by mechanically chopping with blades, followed by Collagenase IV digest (Sigma-Aldrich Cat. No. C5138-1G) in media (DMEM/F12 with 5% FBS, 5µg/mL insulin, and 1% Penstrep/Ampho B) for 45 min at 37°C. Cell suspensions were washed with 2 µg/mL DNAseI for 5 min and further dissociated with 0.05% Trypsin for 10 min. Following a wash with HBBS/2% FBS, cells were passed through a 70µm filter. Lung and primary tumour cells were treated with 1X RBC lysis buffer, followed by resuspension in DMEM/F12 with 10% FBS for FACS. We used the human-specific antibody CD298 (PE, BioLegend, Cat. No. 341704) and the mouse-specific antibody MHCI (APC, ThermoFisher, Scientific Cat. No. 17-5957-80). Flow cytometry was performed using the BD FACSAria Fusion cell sorter. Cell viability was determined by negative staining for SYTOX Blue (ThermoFisher Scientific Cat. No. S34857). Forward scatter area by forward scatter width (FSC W x FSC A) and side scatter area by side scatter width (SSC W x SSC A) was used to discriminate single cells from doublet and multiplet cells. Mouse cells were excluded by gating out CD298-MHCI+. Human primary tumour cells and metastatic cells were selected by gating on Sytox-CD298+MHC-I-. Single cells were sorted directly into each well of a skirted 96-well PCR plate (Fisher Scientific, Eppendorf, Cat. No. E951020443) containing lysis buffer (0.2% Triton X-100, 2 U/µL RNAseOUT, 10µM oligo-dT30VN, and 10µM dNTPs). The plates were snap frozen on dry ice and stored at -80°C until further processing. Total RNA was converted into cDNA using the SmartSeq2 protocol and prepared for Illumina sequencing with the Nextera XT DNA Library Preparation Kit (Illumina, Cat No. FC-131-1096). Cells were sequenced at a depth of 1 million reads/cell on the HiSeq2500.