IntegrateLayers with a reference workflow question

[emiliamoralest]

Hello!
I want to integrate a dataset of Xenium Prime samples to a publicly available reference. All my Xenium samples are stored in layers in one Seurat object, they come from different patients. The reference I want to use is a single cell atlas with 20 different patient samples. My plan is to use IntegrateLayers with RPCA method. I use Seurat 5.2.1. I have a few questions to understand what I should do and the logic behind it:

• Should I integrate the reference alone first?
• Is SCT normalization advised over Log normalization?
• If SCT is better, should I run it both in the reference and in my samples object?
• At what point should I JoinLayers in both objects (or should I leave separated samples)?
• Should I include my reference as a layer in the same object as my samples before integration?

Thank you in advance!