Comprehensive Transcriptomic Analysis of BV2 Microglia Following TDP-43 Knockdown Versus Non-Silencing Control

In this study, we investigate how knockdown of the RNA-binding protein TDP-43 alters the transcriptional program of BV2 microglial cells. Aberrant TDP-43 function is a hallmark of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), and while most research has focused on neurons, emerging evidence points to a critical role for microglia in modulating neuroinflammation and disease progression. We employed RNA interference to specifically deplete TDP-43 in BV2 cells (TDP-43 RNAi) and compared them to cells treated with a non-silencing control siRNA. After 24 hours of knockdown, total RNA was isolated. Libraries were prepared from 500 ng of total RNA using the SEQuoia Complete Stranded RNA Library Prep Kit with ribosomal depletion using the SEQuoia Ribodepletion Kit, capturing both long and short RNA species (mRNAs, tRNAs, and other noncoding transcripts). Libraries were assessed on an Agilent 2100 Bioanalyzer (mean insert size ~300 bp) and quantified on a Quantus fluorometer with the QuantiFluor dsDNA System. Sequencing was performed as single-end 75 bp reads on an Illumina NovaSeq 6000 to a depth of ~50 million reads per library. This workflow allows us to pinpoint pathways and gene networks regulated by TDP-43 in microglia.