Single-Cell RNA-Seq of Tumor Cell Dissociation

The 184-hTERT cells were pelleted and gently resuspended in 200 µl PBS followed by 800 µl 100% methanol and incubation at −20 °C for 30 min to fix, dehydrate and shrink cells. PDX tumour fragments were dissociated into single cells using collagenase/hyaluronidase at 37 °C for 2 h for TNBC tumours or with cold active Bacillus lichenformis (Creative Enzymes NATE0633) in PBS supplemented with 5 mM CaCl₂ and 125 U ml⁻¹ DNAse for HGSC tumours, as described previously^{45 (link)} with additional mechanical dissociation using a gentleMACS dissociator (Miltenyi Biotec). Cells were then pelleted and resuspended in 0.04% BSA/PBS and immediately loaded onto a 10X Genomics Chromium single-cell controller targeting 3,000 cells for recovery. Libraries were prepared according to the 10X Genomics Single Cell 3′ Reagent kit standard protocol. Libraries were then sequenced on an Illumina Nextseq500/550 with 42-bp paired-end reads, or a HiSeq2500 v4 with 125-bp paired-end reads. 10X Genomics Cell Ranger 3.0.2 was used to perform demultiplexing, counting and alignment to GRCh38 and mm10.