# bulk_RNAseq

Ageing RNAseq analysis (OLD vs YOUNG)

"Molecular and phenotypic blueprint of human hematopoiesis links proliferation stress to stem cell aging"
by Emanuele Lettera, et al.

- doi: 10.1084/jem.20251805
- GEO: GSE243327


# RNA-seq analysis.
High quality reads, obtained after reads quality inspection and adapter trimming with Trim Galore (https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/) were aligned to the human reference genome (GENCODE GRCh38 primary assembly) with STAR release 2.7.6a. Gene expression quantification was obtained with featureCounts v2.0.1, using the GENCODE annotation (version v35 primary assembly). Raw counts were normalized by library size, and the differential expression (DE) analysis was performed with the DESeq2 package, testing for the contrast OLD vs. YOUNG. Finally, Benjamini-Hochberg adjusted P values were used to retain significant differentially expressed genes (FDR < 0.05). For the Gene Set Enrichment Analysis (GSEA) genes were ranked by Log-Fold Change and analyzed with the gsePathway function from clusterProfiler (with parameters nPerm = 10000, minGSSize = 120), considering the Reactome database, retaining gene categories with FDR < 0.05. All figures were plotted with ggplot2 in R.
