Using Mis 6th Edition Pdf 43
using mis 6th edition pdf 43
The detailed pipeline v2.0.1 for RNA-seq alignment and splicing analysis is available on _RNAseq.sh. FASTQ files were downloaded from the Gene Expression Omnibus (GEO) database (GSE126543). Adaptors in FASTQ files were removed using trimmomatic (0.39) (ILLUMINACLIP:TruSeq3-PE.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36). The quality of the resulting files was evaluated using FastQC (v0.11.9). RNA-seq reads were mapped to the human (hg38) using STAR v2.7.3a following ENCODE standard options, read counts were generated using RSEM v1.3.1, and differential expression analysis was performed in R v4.0.2 using the DESeq2 package v1.28.140.
RNA-seq densities along the exons were plotted using the sashimi_plot function included in the MISO package (misopy 0.5.4). In the sashimi plot, introns are scaled down by a factor of 15 and exons are scaled down by a factor of 5. RNA-seq read densities across exons are scaled by the number of mapped reads in the sample and are measured in RPKM units. Slight modifications were made to plot_gene.py and plot_settings.py within the sashimi_plot directory of the MISO package to highlight the RNA-seq density plot. The modified sashimi_plot directory is available at ( -43-UNC13A-2021).
We used an induced neuron (iN) system previously established for rapidly differentiating human iPS cells into functional cortical neurons42. In brief, iPS cells (without disease mutation) were cultured using feeder-free conditions on Matrigel (Fisher Scientific CB-40230) using mTeSR1 media (Stemcell Technologies 85850). Cells were transduced with a Tet-On induction system that allows expression of the transcription factor NGN2. Cells were dissociated on day 3 of differentiation and replated on Matrigel-coated tissue culture plates in Neurobasal Medium (Thermo Fisher) containing neurotrophic factors, BDNF and GDNF (R&D Systems) with viral transductions for shScramble or shTDP-43. RNA and protein were extracted 7 days after transduction.
The UNC13A cryptic exon signal was measured using a pair of primers that detect the junction of the CE and the mCherry exon immediately downstream of it. A pair of primers that are mapped within the second exon of eGFP was used to measure the transfection efficiency of the splicing reporter construct, and was used as a normalizer. ΔΔCt was calculated using the cryptic exon signal level in the wild-type HEK 293T cells without TDP-43 overexpression constructs as reference. See Supplementary Table 6 for primers.
The expression levels of the overexpression constructs were measured using a pair of primers that detect the second exon of TDP-43. The primers do detect the endogenous TDP-43 but since the HEK 293T TDP-43 knockout cells do not have TDP-43 expression as shown previously36, using the primers do not interfere with the measurement of the expression levels of TDP-43 constructs in the knockout cells. ΔΔCt was calculated using the TDP-43 expression level in the HEK 293T TDP-43 knockout cells with full length TDP-43 overexpression constructs as reference. RPLP0 and GAPDH were used as internal controls. See Supplementary Table 6 for primers.
Survival curves were compared using the coxph function in the survival (3.1.12) R package, which fits a multivariable Cox proportional hazards model that contains sex, reported genetic mutations and age at onset, and performs a score (log-rank) test. Effect sizes are reported as the hazard ratios. Proportional Hazards assumptions were tested using cox.zph function. The survival curves were plotted using ggsurvplot in suvminer (v.0.4.8) R package. Linear mixed effects models were analysed using lmerTest R package (3.1.3). Statistical analyses were performed using R (version 4.0.0), or Prism 8 (GraphPad), which were also used to generate graphs.
(a) Diagrams showing the design of the UNC13A minigene reporter constructs used to assess the impact of the variants at each locus. The complete design of the reporter construct is shown in Fig. 4d. For clarity, the mCherry and GFP exons that are closest to the promoter (blue in Fig. 4d) are labeled as exon 1, and the downstream exon are labeled as exon 2. REF is the reporter that carries the reference haplotype. M-1 to M-3 carry a single risk variant. M-4 to M-6 carry two risk variants. M-7 carries all three variants, the risk haplotype. (b, c) The locations of the RT-qPCR primer pairs used to detect the inclusion of the cryptic exon (b) and the splicing of EGFP (c, shown in black). (d, e) The expression level of the cryptic exon (d) or the splicing of EGFP (e) in each condition is calculated with reference to the expression level of cryptic exon or the splicing of EGFP from the WT construct in TDP-43-/- HEK-293T cells. The expression of the reporter construct measured using a pair of primers aligned to the second exon of EGFP (c, shown in green) was used to normalize RT-qPCR. The cryptic exon expression levels of each pair of reporters expressed within the same cell line were compared. The splicing of EGFP remained constant across all conditions, verifying equal reporter expression levels and the integrity of the splicing machinery independent of TDP-43.
Counts for specific junctions were tallied by parsing the STAR splice junction output tables using bedtools44. Splice junction parsing pipeline is implemented in Snakemake version 5.5.4 and available at: _parse_star_junctions. Ψ was evaluated using coordinates in Supplementary Table 6:
Cross-linked read files from TDP-43 iCLIP experiments in SH-SY5Y and human neuronal stem cells22 were processed using iCount v2.0.1.dev implemented in Snakemake version 5.5.4, available at _iclip. Sites of cross-linked reads from all replicates were merged into a single file using iCount group command. Significant positions of cross-link read density with respect to the same gene (GENCODE v34 annotations) were then identified using the iCount peaks command with default parameters.
Harmonized summary statistics for the latest ALS GWAS15 were downloaded from the NHGRI-EBI GWAS catalogue54 (accession GCST005647). Locus plots were created using LocusZoom55, using linkage disequilibrium values from the 1000 Genomes European superpopulation56.
Samples were uniformly processed, including adapter trimming with Trimmomatic and alignment to the hg38 genome build using STAR (2.7.2a)38 with indexes from GENCODE v30. Extensive quality control was performed using SAMtools58 and Picard Tools59 to confirm sex and tissue of origin.
The CE was considered detected in a sample if there was at least one uniquely mapped spliced read supporting either the short CE acceptor or the CE donor. As the long CE acceptor was detected consistently in control cerebellum samples, as part of an unannotated cerebellum-enriched 35 bp exon containing a stop codon between exons 20 and 21(Extended Data Fig. 10 a, b), we excluded the long CE acceptor for quantification of UNC13A CE Ψ in patient tissue. Only samples with at least 30 spliced reads at the exon locus were included for correlations. In Fig. 4a, only cortical samples that were concordant for genotypes at rs12973192 and rs12608932, had both STMN2 and UNC13A CE detected, and had at least 30 spliced reads at the exon locus were included in the analysis. Cell-type deconvolution was performed using the top 100 most specific marker genes from neurons, astrocytes, oligodendrocytes, endothelial cells and microglia derived by single-cell RNA sequencing62 with the dtangle63. The NYGC ALS Consortium samples presented in this work were acquired through various IRB protocols from member sites and the Target ALS postmortem tissue core and transferred to the NYGC in accordance with all applicable foreign, domestic, federal, state, and local laws and regulations for processing, sequencing, and analyses. The Biomedical Research Alliance of New York (BRANY) IRB serves as the central ethics oversight body for NYGC ALS Consortium. Ethical approval was given and is effective until 22 August 2022. Informed consent has been obstained from all participants.
Whole-genome sequencing was carried out for all donors, from DNA extracted from blood or brain tissue.Full details of sample preparation and quality control will be published in a future manuscript. In brief, paired-end 150-bp reads were aligned to the GRCh38 human reference using the Burrows-Wheeler Aligner (BWA-MEM v0.7.15)64 and processed using the GATK best-practices workflow. This includes marking of duplicate reads by the use of Picard tools59 (v2.4.1), followed by local realignment around indels, and base quality score recalibration using the Genome Analysis Toolkit65,66 (v3.5). Genotypes for rs12608932 and rs12973192 were then extracted for the samples.
Binding enrichment E-scores were downloaded from Ray et al. (2013)27. Seven-nucleotide sequences that overlapped with either the exonic or intronic SNPs were extracted using a sliding-window approach. Using a custom R script ( _cryptic_splicing/), the average E-scores for each RBP were calculated for each set of 7-mers, and the RBPs were ranked by effect size of the SNPs on average E-score.
An in vivo study using transgenic mice expressing human TDP-43 mutants found that administration of an autophagy-inducing drug could ameliorate TDP-43 pathology in the brain and spinal cord of the transgenic animals . Given the fact that tau and α-synuclein pathologies also implicate disruption of autophagic pathways [172,173,174], developing active pharmacological agents to enhance autophagy flux may alleviate intracellular aggregation-prone proteins. Due to the ubiquitous nature of TDP-43 expression, it may not be a viable therapeutic approach to target TDP-43 in a generalized manner; however, strategies to modify the TDP-43 toxicity and to reduce TDP-43 aggregation may not only benefit FTLD and ALS patients , but also be relevant to more common age-related neurodegenerative disorders such as AD, Lewy body dementia, and LATE.