Integrative Clonal Tracing and CRISPR Screening Identify Temporal and Microenvironmental Determinants in Ovarian Cancer Metastasis

We used scRNA-seq (10x Genomics) coupled with CITE-seq based HTO labeling to profile and compare transcriptomes of omentums and ascital fluid harvested from naive or metastasis bearing wild type C57Bl/6 mice. There are three samples from different animals (M-HTOs) that were pooled for loading onto one 10x Chromium lane + eight samples/M-HTOs pooled for a second lane. Later, three seperate libraries were prepared from each 10x lane: HTO, cell barcode (LARRY), and transcriptome. Within the cell barcode libraries: BC.ID1, BC.ID2, BC.ID3, BC.ID4, BC.ID5 and BC.ID6 are used to distinguish different timepoints of injection - early, mid, and late seeding in descending order (meaning BC.ID6 was earliest timepoint, BC.ID1 is latest timepoint). Overall design: Omentums and lavage/ascital fluid were obtained from naive and metastasis-bearing female C57Bl/6 mice. Omental metastases were established by repeated injection of 400K cell/dose (6x times) of barcoded ID8 p53-/- cells over 12 days, followed by 4 weeks of tumor outgrowth time. We stained each sample with it's own unique hashing antibody so that we could subsequently pool the samples for loading onto two lanes on the 10x Chromium machine and later prepare 2x three libraries consisting of those samples.