GSE129455 - PancreaticDuctalAdenocarcinomaFibroblasts

[ofanobilbao]

Project short name:

PancreaticDuctalAdenocarcinomaFibroblasts

Primary Wrangler:

Ida

Secondary Wrangler:

Associated files

• Google Drive: folder

Link to Ingest

• https://contribute.data.humancellatlas.org/projects/detail?uuid=da9d6f24-3bdf-4eaa-9e3f-f47ce2a65b36

Published study links

• Paper: Cross-Species Single-Cell Analysis of Pancreatic Ductal Adenocarcinoma Reveals Antigen-Presenting Cancer-Associated Fibroblasts

• Accessioned data: GSE129455

Key Events

• [x] Convert published metadata to HCA spreadsheet
• [x] Manually curate dataset to meet HCA metadata standard
• [x] Transfer raw files to ingest to validate data files
• [x] Collect any matrix and cell-type annotation files
• [x] Upload sheet to validate metadata
• [x] Check linking using ingest graph validator
• [x] Ask the Secondary Wrangler for an end-to-end review of the project. Ask the Expertise Wrangler to review specific tabs if needed
• [ ] Submit dataset to Production
• [ ] Complete the Export SOP
• [ ] Convert project data to SCEA format following the SCEA conversion SOP if appropriate

[Wkt8]

Secondary Review Notes:

Add yourself as a curator :)

Should project cell count be reduced to only the mouse cell counts? (11260 cells) As the human cells were not wrangled / represented.

I couldn't see where you got the information for the mouse strain from "C57BL/6J" - can you confirm?

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The enrichment protocol for 'fibroblast_selection' is a bit confusing i think because part of the steps happen for both the 'viable' population and the 'fibroblast-enriched' population? I believe it's the final step which is sorting for DAPI-, CD3- and EpCAM-negative cells that happens only for 'fibroblast-enriched' but the earlier incubation, quenching, spinning, resuspension, etc. happens for both cell populations.

I would recommend one of two options:

1. Include the 'fibroblast_selection' protocol in the process for the 'viable' cell suspensions as well, perhaps renaming it to 'FACS_sorting'
2. Split the 'fibroblast_selection' protocol into three bits:

Staining and selection: After a 3 minute incubation on ice, the ACK was quenched with 2% FBS/PBS. Cells were spun down, resuspended in PBS supplemented with 0.5% BSA and 2 mM EDTA and blocked with anti-mouse CD16/CD32 Fc Block for 15 minutes at 4°C. Staining of mouse tissue for flow sorting was performed with the following antibodies: CD45-AF647, CD31-AF647, and EpCAM-AF647, for 30 minutes at 4°C. Prior to sorting, DAPI was added to the cell suspension. Cells were sorted using a BD FACSAria Fusion instrument.

Viable_selection Following exclusion of debris, 50,000-100,000 DAPI-negative cells were collected from each sample (for ‘all viable cell’ fraction).

fibroblast_selection Then sorting for DAPI-, CD45-, CD31- and EpCAM-negative cells was performed (for ‘fibroblast-enriched’ fraction). Both fractions were collected in 20% FBS/PBS.

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I think it should be CellRanger (v1.3) that was actually used for creating fastqs, although 1.2.0 was used for aligning but.. either should be fine as that's a value judgement there.

Computational_method is 'Cellranger ;' can remove the semi-colon?

[idazucchi]

I've updated the cell count to reflect only the mouse cells sequenced The mouse strain comes from GEO samples

Enrichment protocol I've split the enrichment protocol in two:

1. viable cells : Preparation for FACS (quenching + blocking) + DAPI staining and FACS sorting
2. fibroblasts : antibodies staining and FACS sorting

The viable cells is applied to all cell suspensions, fibroblasts only to the four fibroblasts cell suspensions

[idazucchi]

exported without issue and filled out the import form