Comprehensive Transcriptomic Analysis of BV2 Microglia Following TDP-43 Knockdown Versus Non-Silencing Control
Comprehensive Transcriptomic Analysis of BV2 Microglia Following TDP-43 Knockdown Versus Non-Silencing Control
Twenty-four hours after transfection with TDP-43 siRNA or non-silencing control siRNA, culture medium was gently aspirated and the cells washed once with 1 mL of ice-cold, RNase-free PBS. After complete removal of PBS, 1 mL of TRIzol Reagent (Life Technologies: 15596018) was added directly to each well. Plates were placed on a rotary shaker at room temperature (∼22 °C) and agitated at 300 RPM for 5 minutes to ensure thorough cell lysis. Lysates were then transferred to pre-chilled 1.5 mL microcentrifuge tubes for downstream RNA extraction. BV2 murine microglial cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM; Sigma-Aldrich D5796) supplemented with 10 % heat-inactivated fetal bovine serum (Gibco 10500064) and 1 % penicillin–streptomycin (Gibco 15140122) at 37 °C in a humidified 5 % CO₂ incubator. For routine passaging, cells were split every 2–3 days when they reached ~70–80 % confluency: spent medium was aspirated, the monolayer washed once with sterile PBS, and cells detached by a 2–3 min incubation with 0.05 % trypsin-EDTA (Gibco 25300054) at 37 °C. Trypsin was quenched with complete medium, cells were collected by centrifugation at 300 × g for 5 min, resuspended in fresh DMEM, and reseeded at a 1:5 split ratio. Twenty-four hours before experimental manipulation (siRNA transfection), BV2 cells were seeded into 6-well plates at approximately 60 % confluency (∼2 × 10⁵ cells per well) to ensure uniform density and optimal growth conditions.   BV2 microglial cells at ~60 % confluency (seeded 24 h prior at ~2 × 10^5 cells per well in 6-well plates) were transfected with siRNA using Lipofectamine RNAiMAX (Invitrogen 13778075) according to the manufacturer's instructions. Briefly, 20 pmol of either TDP-43 siRNA or non-silencing control siRNA (Qiagen) was diluted in 125 µL of Opti-MEM® (Gibco) and, in a separate tube, 3 µL of RNAiMAX reagent was diluted in 125 µL of Opti-MEM®. After a 5 min room-temperature incubation, the diluted siRNA and RNAiMAX solutions were combined and incubated for 10 min to allow complex formation. The 250 µL siRNA–lipid complexes were then gently added dropwise to each well containing 2 mL of fresh complete DMEM. Cells were returned to the 37 °C, 5 % CO₂ incubator and incubated for 6 h, after which the medium was replaced with 2 mL of fresh DMEM. Transfection efficiency and cell morphology were monitored by light microscopy, and cells were harvested for RNA extraction 24 h post-transfection. Total RNA was extracted from BV2 cells 24 hours post-transfection using a modified TRIzol® protocol to maximize yield and purity. After aspirating culture medium and washing once with 1 mL of ice-cold, RNase-free PBS, 1 mL of TRIzol Reagent (Life Technologies: 15596018) was added directly to each well; plates were agitated at 300 RPM for 5 minutes at room temperature to ensure complete lysis. Lysates were transferred to pre-chilled 1.5 mL tubes, incubated 5 minutes at ambient temperature, then supplemented with 200 µL of chloroform. Tubes were vortexed vigorously for 15 seconds and incubated 3 minutes at room temperature before centrifugation at 12,000 × g for 15 minutes at 4 °C. The upper aqueous phase (\~400 µL) was carefully pipetted into a new tube, mixed with an equal volume of chloroform, and centrifuged again under the same conditions to further remove phenol contaminants. The resulting aqueous phase (\~400 µL) was then combined with 500 µL of isopropanol, gently inverted to precipitate RNA, and incubated 10 minutes at room temperature. Following centrifugation at 12,000 × g for 10 minutes at 4 °C, the supernatant was discarded and the RNA pellet washed twice with 1 mL of 70% ethanol (centrifuging at 7,500 × g for 5 minutes at 4 °C between washes). Pellets were air-dried (∼5–10 minutes) until residual ethanol evaporated, then dissolved in 30 µL of DEPC-treated water by gentle pipetting and incubating 5 minutes at 55 °C. RNA concentration and purity (A260/A280 and A260/A230 ratios) were measured on a NanoDrop spectrophotometer, and integrity was confirmed on an Agilent 2100 Bioanalyzer using the RNA 6000 Nano Kit; only samples with RIN ≥ 9.0 proceeded to library preparation.  Library preparation was performed on 500 ng of total RNA per sample using the SEQuoia Complete Stranded RNA Library Prep Kit (Bio-Rad, Cat. No. 17005726) with ribosomal RNA removal achieved via the SEQuoia Ribodepletion Kit (Bio-Rad, Cat. No. 17006487). In brief, each RNA sample was first incubated with ribodepletion reagents to deplete rRNA species, then the remaining RNA was fragmented enzymatically to an average size of ~200 nt. Fragment ends were repaired and phosphorylated, and a single adenosine residue was added to each 3′ end to facilitate directional adapter ligation. Strand‐specific adapters, containing unique molecular identifiers and flow-cell binding sites, were ligated to both ends of the fragments. Adapter‐ligated RNA was reverse‐transcribed to first-strand cDNA, followed by second-strand synthesis to generate double-stranded libraries. Libraries were enriched by 12 cycles of PCR using high-fidelity polymerase, during which index sequences were incorporated. Amplified libraries were purified with magnetic beads to remove primer dimers and small fragments, then assessed for size distribution on an Agilent 2100 Bioanalyzer (DNA High Sensitivity chip), confirming a mean insert size of ~300 bp. Final library concentrations were measured on a Quantus fluorometer using the QuantiFluor dsDNA System (Promega) before pooling and sequencing.