Single-Cell Rna-Seq Of Breast Cancer Infiltrating T Cells (Case 1)

Single cell suspensions were generated from two individual TNBC primary tumor samples (this entry contains case 2) and the viable cells were FACS sorted for CD3⁺ T cells. Sorted cells were then counted and assessed for viability. Single cell library preparation was carried out as per the 10X Genomics Chromium Single cell protocol for the v2 reagent kit (10X Genomics, Pleasanton, CA, USA). Cell suspensions were loaded onto a Chromium Single Cell Chip along with the reverse transcription (RT) mastermix and single cell 3’ gel beads. Following generation of single cell gel bead-in-emulsions (GEMs), reverse transcription was performed using a C1000 Touch Thermal Cycler with a Deep Well Reaction Module (Bio-Rad Laboratories, Hercules, CA, USA). Amplified cDNA was purified using SPRIselect beads (Beckman Coulter, Lane Cove, NSW, Australia) and sheared to approximately 200bp with a Covaris S2 instrument (Covaris, Woburn, MA, USA) using the manufacturer’s recommended parameters. Sequencing libraries were generated with unique sample indices (SI) for each sample. Libraries were sequenced on an Illumina HiSeq 2500 High Output Mode using V4 clustering and sequencing chemistry. This dataset contains the raw .bcl files.