Recommended workflow to pre-process and demultiplex scRNAseq data based on the HTOs and ADTs

Question

[mete.han.celebi]

Hello everyone,

Soon, I will receive a data from a study where the tumor cells are harvested from 9 animals and afterwards each biological sample is labeled with a unique TotalSeq-C hashtags to be able to track the data back to the biological replicate. After that step, cells are sorted as CD45+ and CD45-. Then only CD45+ cells are additionally stained with TotalSeq-C mouse universal cocktail that targets immune cells receptors. Therefore, the data that I will receive to analyze will have both gene expression, ADT, and HTO libraries and this data is generated by 10x Genomics NEXT GEM Flex kit. I need to identify which cell barcode came from which mouse based on the HTOs. Then, I need to identify which cell barcode came from which immune cell that is labeled with universal cocktail.

In this experiment, during the library prep step 4 well of the chromium controller is used therefore I will have 4 different sets of Fastq files.

My question is, should I run cell ranger multi first by specifying the HTO sequences ( I have 9 unique sequences ) ? If so, after that step what sort of workflow should I follow to assign the mouse origin and immune cell type origins based on the HTOs and ADTs?

I would be so happy to hear and learn from you!

Thank you! Metehan

Answer 1

[GenoMax]

While we wait for more detailed answers, in case you have not seen this: https://www.10xgenomics.com/support/software/cell-ranger/latest/analysis/running-pipelines/cr-3p-multi

[mete.han.celebi]

Thank you very much! Yes I haven't seen this. That is very valuable!