Hi

I was hoping someone might be able to help answer a question regarding subsetting of scRNAseq data and processing following this for seuratV5 (version: 5.1.0).

I have integrated my data by treatment and have an integrated.cca reduction within the seurat object.

This creates this object below.

Following this I go onto to subset out some clusters. People recommend you then renormalise the data and refind variable features then re-cluster the data. This works in seuratV4 using as below.

Is this the correct way to go about this?

When I do this it seems to lose the integration or lose the subsetting.

Would people recommend I should be re-integrating the data or not rerunning the PCA? or setting a different assay for the PCA (as the only assay in seuratV5 in my object is RNA I can't work out how to do this). Thanks for the help!