The steps are basically: (1) prepare the GEO HTS metadata spreadsheet, (2) upload files to GEO FTP, (3) submit the metadata file via the GEO web form, and (4) respond to any curator questions (esp. raw FASTQ).

1) Make a single dataset folder name for GEO (recommended)

GEO wants one folder per submission/dataset. Create one top-level folder name that will be uploaded as-is:

cd ~/Downloads
mv GEO_submissions GeoMx_DSP_M666_UKE_Hamburg

Inside it should remain:

• dcc/
• pkc/
• annotation/
• README.txt
• counts_matrix.tsv

2) Prepare the GEO HTS metadata spreadsheet (REQUIRED)

On the GEO “Submit HTS” page:

• Download the HTS metadata spreadsheet template.
• Fill at least these tabs:

STUDY tab

• Title, summary, overall design.

• Add the “raw FASTQ missing” sentence if you still don’t have FASTQs:

  Raw sequencing FASTQ files are currently not available to the authors; processed DCC outputs and PKC probe metadata are provided. FASTQs will be deposited to SRA and linked once obtained.

SAMPLES tab (most important)

One row per ROI/AOI (so ~45 rows for your DCCs).

• Use Sample_ID (from your Sheet8/clean TSV) as the sample/library name.
• For each row, set processed data file to the matching DCC filename (e.g., DSP-...-A02.dcc).
• Include key sample characteristics (Group/Location/PCR/Case/etc.) from your annotation.

PROTOCOLS tab

• Sample prep + GeoMx DSP workflow description.
• Mention panels: Hs_R_NGS_WTA_v1.0, GeoMx_Hs_CTA_v1.0, GeoMx_COVID19_v1.0.
• State: DCC exported from NanoString GeoMx NGS pipeline; PKC required to interpret RTS_IDs.

FILES / supplementary (if the template has it)

List:

• all DCCs
• all PKCs
• the annotation file(s) (at least the clean TSV or the Excel)
• README.txt
• counts_matrix.tsv (optional, but you have it already)

Tip: In the metadata, reference counts_matrix.tsv as “gene-level raw counts matrix (targets x ROIs)” and keep DCCs as the authoritative per-ROI outputs.

3) FTP upload to GEO (data files only)

1. Connect to GEO’s FTP using the credentials/path GEO gives you (your personal upload directory).

2. In that upload directory, create a dataset folder, e.g.:

   • GeoMx_DSP_M666_UKE_Hamburg/

3. Upload only the data files by FTP:

   • the whole folder with subfolders: dcc/, pkc/, annotation/, plus README.txt, counts_matrix.tsv
4. Do NOT upload the metadata spreadsheet by FTP (GEO explicitly says upload metadata via the web form).

4) Submit the metadata spreadsheet on the GEO web page

After the FTP transfer finishes:

• Go to Submit a new high-throughput sequencing submission
• Upload the metadata spreadsheet file for this dataset
• Submit → this places it in the GEO processing queue.

5) If GEO asks for raw FASTQs

This is the most common friction point for GeoMx when FASTQs aren’t available.

• If they allow: they’ll accept processed now and ask you to link SRA later.
• If they require: you’ll need to obtain FASTQs from the provider or email GEO and explain you only received DCC/PKC and are requesting FASTQs.

TODO: upload/paste the GEO HTS metadata spreadsheet template (blank) or a screenshot of the SAMPLES tab columns — to have a look which columns to fill with which fields from your annotation/M666_Sheet8_annotation_clean.tsv.