README.md

# Vavassori_LNP2022_RNAseq_electro

Vavassori V, Ferrari S, Beretta S, et al.
**Lipid nanoparticles improve ex vivo gene editing of human hematopoietic cells.**
2022.

Whole transcriptomic analyses on human CD4+ T cells from 3 healthy donors, using a previously optimized culture/editing protocol and Cas9 reagents targeting the first intron of CD40LG gene. We compared untreated (UT) cells, mock electroporated cells (mock electro), cells electroporated with RNP without (RNP) or with AAV6 donor (RNP+AAV6), and AAV6-transduced cells (AAV6 only - no electroporation) derived from 3 healthy donors 12 hours after treatment, and at long term.

- GEO: [GSE216248](https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE216248)

---

### Analyses ###

Stanrdard RNA-seq pipeline on 33 samples: 3 UT at day 3, 15 samples at day 4, and 15 samples at day 12.<br>
In details, for both time points (i.e., day 4 and day 12) DGE analysis on samples was performed with DESeq2, comparing dfferent groups:
```
results(DESeq.ds, pAdjustMethod = "BH", independentFiltering = TRUE, contrast = c("groups", Group1, Group2), alpha = 0.05)
```
Enrichment on (positive or negative) differentially expressed genes (FDR < 0.05) and GSEA on pre-ranked gene lists (ordered by log2 Fold Change values) agasint the MSigDB Hallmark database were performed with ClusterProfiler:
```
enricher(PosDEGs, universe = GeneUniverse, pvalueCutoff = 0.05, TERM2GENE = HallMarkDB)

GSEA(FullGenesOrderLogFC, TERM2GENE = HallMarkDB, nPerm = 10000, pvalueCutoff = 0.05)
```

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### Directories and Files ###

- _paper_plots.R_: script used to produce the plots of the paper;
- _paper_data_: folder with data used to produce the plots of the paper;
- _GEO_RNAseq_electro.xlsx_: GEO template file;
- _config.yaml_: config files of the RNA-seq analysis pipeline.