Feb 03, 2025
Bulk RNA-seq and data analysis
- 1Institut Imagine
- Team Deleidi
Protocol Citation: Maria Jose Perez J. 2025. Bulk RNA-seq and data analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov1d46pvr2/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 26, 2024
Last Modified: February 03, 2025
Protocol Integer ID: 112821
Funders Acknowledgements:
ASAP
Abstract
Bulk RNA-seq and data analysis
Bulk RNA-seq and data analysis
Bulk RNA-seq and data analysis
Isolate total RNA from three biological replicates per sample of iPSC-derived neurons (treated for 24 hours), astrocytes, and microglia (treated for 6 hours) using the RNeasy Mini Kit (Cat. number 74106, QIAGEN, Germany) according to the manufacturer’s instructions.
Enrich mRNA via poly(A) selection. (Done by GENEWIZ GmbH- Leipzig, Germany)
Prepare cDNA libraries using strand-specific RNA-seq with poly(A) selection (Illumina, CA, USA). (Done by GENEWIZ GmbH- Leipzig, Germany)
Perform sequencing on the Illumina NovaSeq platform (Illumina, CA, USA) to generate 150 bp paired-end reads.(Done by GENEWIZ GmbH- Leipzig, Germany)
Perform differential gene expression analysis with DESeq2 (version 1.44.0, RRID: SCR_015687) to normalize counts and identify differentially expressed genes (DEGs)
Conduct functional enrichment analysis of DEGs (fold change > 2; Benjamini‒Hochberg FDR < 0.05) using ShinyGO (version 0.80, http://bioinformatics.sdstate.edu/go/, RRID: SCR_019213).
Filter data to assess statistical significance (FDR < 0.05).
Generate Venn diagrams of DEGs shared between iPSC-derived neurons, astrocytes, and microglia using Draw Venn Diagram (https://bioinformatics.psb.ugent.be/webtools/Venn/).
Data of the study were deposited into GEO under accession number GSE274283.