title: Comprehensive Transcriptomic Analysis of NSC34 Motor Neurons following TDP43 Modulation: Knockdown vs. Control and Wild Type vs. TDP43 M337V Mutation
design: Comprehensive Transcriptomic Analysis of NSC34 Motor Neurons following TDP43 Modulation: Knockdown vs. Control and Wild Type vs. TDP43 M337V Mutation
library_name: TDP43-Mutant 2_s
library_construction_protocol: For RNA extraction, the initial step involved the removal of cell culture media, followed by a single wash of cells with ice-cold PBS. Subsequently, 1 ml of Trizol per sample was added after complete removal of PBS, and the cells were subjected to shaking for 5 minutes at room temperature on a rotary shaker at 300 RPM.  The NSC34 motor neuron cell line, initially at passage 3 (p3), was obtained from Tebubio, with a primary focus on these cells. All cells, including NSC34, were cultured and sustained in complete media (CM) in a T75 flask, and subculturing was performed upon reaching 80-90% confluency. The complete media (CM) consisted of Dulbecco's Modified Eagle's Medium (DMEM Sigma-Aldrich: D5796) containing 4500 mg/L glucose, L-glutamine, and sodium bicarbonate, excluding sodium pyruvate. This liquid medium was sterile-filtered and enriched with 10% Fetal Bovine Serum (FBS, Gibco: 10500064) and 1% Penicillin-Streptomycin (Gibco: 15140122) to support cell maintenance and growth.The subculturing process involved rinsing cells with Dulbecco's Phosphate-Buffered Saline (DPBS, Gibco: 14190-144) and dissociating them using TrypLE™ Express Enzyme (Gibco: 12604013). Subsequently, the dissociated cells were harvested, centrifuged at room temperature at 120 revolutions per minute (rpm) for 5 minutes, the supernatant was discarded, and the cells were reconstituted with complete DMEM medium. These reconstituted cells were then maintained under the aforementioned culture conditions. For RNA interference (RNAi) targeting TDP43 in NSC34 motor neurons and other cell lines, Lipofectamine™ RNAiMAX Transfection Reagent was utilized to transfect cells with two TDP43-specific siRNAs: Mm_Tardp 1 siRNA and Mm_Tardp 2 siRNA. These siRNAs targeted the TDP43 gene with sequences AACGATGAACCCATTGAAATA and CAGTAACATGGTAACATTAAA, respectively. Non-silencing Qiagen AllStars Negative Control siRNA and AllStars Mm/Rn Cell Death Control siRNA served as controls for non-specific effects and cell viability. Cells were seeded 24 hours prior to transfection, reaching 45-60% confluency in various plate formats. On the day of transfection, a siRNA-lipid complex was formed by combining Lipofectamine RNAiMAX Reagent and siRNA diluted in Opti-MEM Medium. The complex was added to fresh antibiotic-free media, resulting in a final siRNA concentration of 20nM. Cells were incubated for 6 hours, media changed, and further incubated for up to 24 hours. Transfection efficiency and TDP43 knockdown were assessed using Cell Death Control siRNA, qPCR, and western blot techniques.  The TDP43 Wild-Type and Mutation Plasmid construction involved synthesizing the human TDP43 open reading frame in the pcDNA3.1+C-eGFP vector, resulting in the Cloning:TDP43_WTT_pcDNA3.1(+)-C-eGFP plasmid. Three ALS-linked TDP43 mutations (Q331K, M337V, and G294A) were incorporated using site-directed mutagenesis and cloned into the same vector, resulting in mutant plasmids. Synthesis included sequence design, PCR amplification, ligation, bacterial screening, and sequencing for quality control. Plasmids underwent transformation, mini-prep, and extraction using the Zyppy™ Plasmid Miniprep Kit. Quality checks were performed using Nanodrop, and sequencing by Genomed confirmed sequence accuracy. Sequenced plasmids were used for maxi-preps to ensure high concentrations, and sequencing results were verified for mutation presence.  For TDP43 wildtype and mutant expression in cells, plasmids were individually transfected into cells 24 hours after seeding, employing Lipofectamine™ 3000 Transfection Reagent. Cells were grouped for transfection with wildtype (WTT), mutants (QKT, MVT, GAT), and eGFP (transfection control). Transfection efficiency and TDP43 expression were assessed by observing green fluorescence and confirmed through qPCR and western blot techniques after 48 hours of incubation under normal conditions. The resulting cell lysate underwent vigorous pipetting and vortexing. The homogenized sample was then incubated for 5 minutes at room temperature to facilitate the complete dissociation of nucleoprotein complexes. In the next step, 0.2 ml of chloroform per 1 ml of Trizol reagent was added, vigorously vortexed until the lysate became visibly white, and incubated at room temperature for 2 to 3 minutes. Following centrifugation at 12,000g for 15 minutes at 2-8°C, the upper aqueous phase was carefully transferred to a fresh tube without disturbing the interphase. A volume of 400 µl of the aqueous phase was collected, and an equal volume of chloroform was added. After another centrifugation at 12,000g for 15 minutes at 2-8°C, 0.5 ml of isopropyl alcohol was added to the samples, mixed, and incubated at room temperature for 10 minutes. To enhance visibility, 10 µl of glycogen was introduced. Subsequently, the samples were centrifuged at 12,000g for 10 minutes at 2-4°C. Following removal of the supernatant, the RNA pellet underwent two washes with 1 ml of 70% ethanol, accompanied by vortexing and centrifugation at 7,500g for 5 minutes. The pellet was air-dried for 30 minutes, and the RNA was resuspended in 30 µl of DEPC-treated water. The quantification of RNA was carried out using a Nanodrop Spectrophotometer. The RNA specimens, encompassing those derived from RNAi investigations and TDP43 mutation experiments, were readied for sequencing subsequent to the RNA extraction process. Samples attaining RNA Integrity Number (RIN) values of 8.5 or higher, as ascertained through bioanalyzer analysis, were chosen for further examination. Library construction was executed employing the SEQuoia Complete Stranded RNA Library Prep Kit (Bio-Rad: 17005710), intentionally crafted for the comprehensive capture of both lengthy and concise RNA molecules. This kit facilitates an exhaustive portrayal of the coding and noncoding transcriptome in a singular library. Subsequent to library preparation, ribodepletion was implemented utilizing the SEQuoia RiboDepletion Kit (Bio-Rad: 17006487). This phase proficiently eliminated fragments of ribosomal and mitochondrial RNA, thereby preserving elusive transcripts that are commonly forfeited
identifiers.namespace: European Bioinformatics Institute
identifiers.id: E-MTAB-13738:TDP43-Mutant 2_s