Hi everyone,

In the next journal club, we will try to navigate a brand-new single-cell paper in HIV research (publication date: May 2025!).

Since the paper is long and has some complex passages, we will not go through the entire paper. Instead, the purpose of the journal club will be to illustrate (and hopefully discuss) some key analysis common to single cell papers and discuss the major findings and implications – mainly in the CD4 compartment.

The main questions are:

1. What did they find?
2. How did they use single-cell analysis to achieve their results (which analysis supports which conclusion)?
3. How relevant is this knowledge for the field/future studies?

Here is a detailed reading guide/prioritized reading checklist:

• Introduction and Discussion
• Who does the term Immunological non-responders refer to
• What characterizes immunological non-responders
• What is the aim of the study – compare with outcomes reported in Discussion
• Methods
• Study design: who was included, and which are the data sources?
• How were samples treated
• Other methods that will be discussed (you may read these if you are interested – but you do not need to)
• Sections scRNA-library preparation, Differential gene analysis, gene set enrichment analysis, gene set scoring algorithm, RNA velocity and pseudotime, bulk RNA-seq
• Results
• What is a single-cell atlas?
• What do differentially expressed genes tell us – here and in general? Which populations were compared?
• How do you interpret enrichment analysis results (GO, GSEA)?
• We will focus on the differences between these approaches at the journal club – for now, just think about which message you get, with your current knowledge, when you see these results
• Can we trust the results from the pDC analysis?
• Get an overview of the CD4 cluster differences between IR and INR; do not focus too much on single genes
• How do you interpret the results of trajectory analysis
• We will focus on this approach at the journal club – for now just think about which message you get, with your current knowledge, when you see these results
• How are these results validated?
• Figures
• Figure 1: what does the heatmap tell you?
• Figure 2: focus on b, c, d (what does the volcano tell you?), f, g, h
• Figure 3: which are the most informative panels to understand the main findings?
• Figure 4: focus on panel a, b, g (what does it tell you?) i&j (what do you get from them at first sight?)

I attach a copy of the paper where non-relevant sections are crossed out and a clean version (if you find the crosses annoying).

I know that single-cell papers can be complex. Still, with the increasing prevalence of single-cell studies, it is important to understand how to identify the main findings, quickly scan the most important figures, and know the major pitfalls. I hope the questions above help you navigate the paper and focus your attention.

If you have any questions or issues with the attachments, please just ask.

Best,

Giacomo