{"doi":"10.17504/protocols.io.bhbkj2kw","title":"HTAPP_Processing human ovarian cancer ascites to a single-cell suspension for single-cell RNA-seq v2","abstract":"<p>This protocol is used to process human ovarian cancer ascites to a single-cell suspension compatible with droplet-based single-cell RNA-Seq technology.  A majority of ascites samples have a high proportion (&gt;90%) of CD45-expressing (CD45+) cells, thus greatly reducing the ability to capture non-immune and malignant cells. To address this issue, we have included a CD45+ cell depletion step to reduce the number of CD45+ cells.In such samples, where the proportion of immune cells is very high, a large proportion of both CD45+ and CD45- cells can be recovered post-depletion, allowingsimultaneous assessment of the immune and non-immune microenvironment. In cases with a lower initial proportion of CD45+ cells, however, non-immune cells tend to be dominant following CD45+ cell depletion.  Of note, many ascites samples contain multicellular aggregates, or spheroids, which pose challenges during sample processing and dissociation. Larger aggregates are removed through an initial 70 μm straining (Step 14) whereas smaller residual aggregates, when present, can be removed through a final 30 μm straining (optional Step 20). Yet, we have encountered instances where small aggregates cannot be efficiently removed through straining. This protocol is not optimized for those cases. In addition, we would like to note that ascites can be quite variable and further optimization of this protocol may be needed to handle cases with atypical characteristics.  For the Human Tumor Atlas Pilot Project (HTAPP), this protocol was successfully applied to 4 ovarian cancer ascitessamples.   Description of this protocol and guidance for testing and selecting methods for processing other tumor and sample types can be found inSlyper et al.</p>","journal":null,"year":2020,"id":4949,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":0,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.0619,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2020-06-09","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":5595,"name":"Parin Shah","orcid":"0000-0003-4648-7830","position":1,"is_corresponding":false},{"id":3723,"name":"Mei-Ju Su","orcid":null,"position":2,"is_corresponding":false},{"id":3722,"name":"Isaac Wakiro","orcid":"0000-0002-5248-4823","position":3,"is_corresponding":false},{"id":11837,"name":"Sara Napolitano","orcid":"0000-0002-9453-8586","position":4,"is_corresponding":false},{"id":11838,"name":"Jingyi Wu","orcid":"0000-0003-0047-6378","position":5,"is_corresponding":false},{"id":11839,"name":"Sébastien Vigneau","orcid":"0000-0002-9091-2067","position":6,"is_corresponding":false},{"id":3727,"name":"Asaf Rotem","orcid":"0000-0002-6859-7435","position":7,"is_corresponding":false},{"id":2697,"name":"Orit Rozenblatt–Rosen","orcid":"0000-0001-6313-3570","position":8,"is_corresponding":false},{"id":29633,"name":"Prisca Liberali","orcid":"0000-0003-0695-6081","position":10,"is_corresponding":false},{"id":3735,"name":"Orit Rozenblatt‐Rosen","orcid":null,"position":11,"is_corresponding":false},{"id":5601,"name":"Bruce E. Johnson","orcid":"0000-0002-5252-3677","position":12,"is_corresponding":false},{"id":5593,"name":"Benjamin Izar","orcid":"0000-0003-2379-6702","position":0,"is_corresponding":true}],"reference_count":0,"raw_metadata":null,"created_at":"2026-03-01T18:20:47.508186Z","pmid":null,"pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}