Journal: Data in Brief

Article Title: Data of RNA-seq transcriptomes of liver, bone, heart, kidney and blood in klotho mice at a pre-symptomatic state and the effect of a traditional Japanese multi-herbal medicine, juzentaihoto

doi: 10.1016/j.dib.2022.108197

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Article Snippet: How data were acquired , DNA sequencing Instrument: NovaSeq 6000 (Illumina) Program: NovaSeq Control Software v1.3.0 (Illumina) Library construction reagents: TruSeq Stranded mRNA Sample Prep Kit (illumina) Sequencing reagents: NovaSeq 6000 S4 Reagent Kit (illumina) Real-time PCR Instrument: StepOnePlus (Thermofisher) Reagent: Power SYBR® Green RNA-to-C T 1-Step Kit (Thermofisher) Western Blot Homogenization: disposable homogenizer (Nippi BioMasher) Protein assay reagents: BCA protein assay (Thermo Fisher Scientific), ECL Prime Western Blotting Detection Reagents (GE Healthcare) Electrophoresis: myPower II 300 (ATTO) Detection program: ImageJ (NIH), ChemiDoc system (Bio-Rad).

Techniques: Alternative Splicing, DNA Sequencing, Control, Software, Sample Prep, Illumina Sequencing, Real-time Polymerase Chain Reaction, SYBR Green Assay, Western Blot, Homogenization, Bicinchoninic Acid Protein Assay, Electrophoresis, Knock-Out, Sequencing, Marker

Journal: bioRxiv

Article Title: A nuclear RNA degradation code for eukaryotic transcriptome surveillance

doi: 10.1101/2024.07.23.604837

Figure Lengend Snippet: A , Western blot analysis of knockdown samples prior to PAS-seq to confirm depletion of EXOSC3, MTR4, ZFC3H1, or PABPN1. Tubulin and GAPDH were used as loading controls. B , Schematic depicting the synthesis of IPA transcripts and three common examples of non-IPA transcripts. IPA transcripts are produced when a pre-mRNA is cleaved and polyadenylated within the intron, leaving an intact 5′ ss upstream of the poly(A) tail. For non-IPA transcripts, cleavage and polyadenylation may occur within the terminal exon or within an alternative last exon following alternative splicing. C , Stacked barplots depicting the frequency of PAS position across all upregulated transcripts (log 2 FC > 1, FDR ≤ 0.05) in HEK293T cells depleted of EXOSC3, MTR4, ZFC3H1, or PABPN1.

Article Snippet: Anti-FLAG mouse (Sigma-Aldrich, F1804-200UG) Anti-beta Tubulin (Invitrogen, PA1-16947) Anti-GAPDH (Santa Cruz, sc-365062) Anti-ZFC3H1 (CCDC131) (Bethyl, A301-456A) Anti-MTR4 (Abcam, AB70551) Anti-PABPN1 (Bethyl, A303-524A) Anti-ZCCHC8 (Bethyl, A301-806A) Anti-EXOSC3 (Proteintech, 15062-1-AP) Anti-U1-70K (Millipore, clone 964.1, 05-1588) Anti-U1A (Santa Cruz, sc-101149) Anti-U1C (Sigma, SAB4200188-200UL) Anti-ARS2 (Bethyl, A304-550A) Anti-CPSF100 (Bethyl, A301-581A) Anti-CPSF73 (Bethyl, A301-091A) Anti-CPSF30 (Bethyl, A301-585A) Anti-FLAG rabbit (Cell Signaling, 14793S) Anti-SC-35* (SRRM2) (Abcam, ab11826) *Recently, a study reported that anti-SC35, a widely used marker of nuclear speckles, recognizes SRRM2

Techniques: Western Blot, Knockdown, Produced, Alternative Splicing

Journal: bioRxiv

Article Title: A nuclear RNA degradation code for eukaryotic transcriptome surveillance

doi: 10.1101/2024.07.23.604837

Figure Lengend Snippet: A , Density plots of the log 2 FC for all IPA or non-IPA transcripts following depletion of EXOSC3, MTR4, ZFC3H1, or PABPN1 in wild-type HEK293T cells. IPA transcripts are depicted in red and non-IPA transcripts are depicted in grey. A positive log 2 FC indicates that the transcript is upregulated upon knockdown. B & C , PAS-seq data tracks for the genes TP53 ( B ) and RNF166 ( C ) following depletion of EXOSC3, MTR4, ZFC3H1, or PABPN1 by RNAi in wild-type HEK293T cells. Each peak represents reads mapped to the 3′ end of mRNAs. D , Heatmap of the log 2 FC measured for all IPA transcripts that were found to be upregulated in at least one knockdown condition (log 2 FC > 1, FDR ≤ 0.05). A positive log 2 FC indicates that the IPA transcript is upregulated upon knockdown. E , Box plot depicting the strength of the nearest upstream 5′ ss as measured by MaxEnt. PAXT-regulated: Significantly upregulated IPA transcripts (log 2 FC > 1, FDR ≤ 0.05, N = 507) following depletion of EXOSC3, MTR4, and ZFC3H1. Stable IPA transcripts: IPA transcripts whose expression did not significantly change following depletion of EXOSC3, MTR4, and ZFC3H1 (Counts per million (CPM) in control cells > 1, log 2 FC < 1 and log 2 FC > -1, FDR > 0.05, N = 687). Statistical analysis was calculated by Mann-Whitney test.

Article Snippet: Anti-FLAG mouse (Sigma-Aldrich, F1804-200UG) Anti-beta Tubulin (Invitrogen, PA1-16947) Anti-GAPDH (Santa Cruz, sc-365062) Anti-ZFC3H1 (CCDC131) (Bethyl, A301-456A) Anti-MTR4 (Abcam, AB70551) Anti-PABPN1 (Bethyl, A303-524A) Anti-ZCCHC8 (Bethyl, A301-806A) Anti-EXOSC3 (Proteintech, 15062-1-AP) Anti-U1-70K (Millipore, clone 964.1, 05-1588) Anti-U1A (Santa Cruz, sc-101149) Anti-U1C (Sigma, SAB4200188-200UL) Anti-ARS2 (Bethyl, A304-550A) Anti-CPSF100 (Bethyl, A301-581A) Anti-CPSF73 (Bethyl, A301-091A) Anti-CPSF30 (Bethyl, A301-585A) Anti-FLAG rabbit (Cell Signaling, 14793S) Anti-SC-35* (SRRM2) (Abcam, ab11826) *Recently, a study reported that anti-SC35, a widely used marker of nuclear speckles, recognizes SRRM2

Techniques: Knockdown, Expressing, Control, MANN-WHITNEY

Journal: bioRxiv

Article Title: A nuclear RNA degradation code for eukaryotic transcriptome surveillance

doi: 10.1101/2024.07.23.604837

Figure Lengend Snippet: A , Schematic of the mRNA structure of the eGFP reporters used in B - F . The reporters contain the coding sequence for eGFP followed by either a wild-type (WT) or mutant (Mut) 5′ ss in the 3′ UTR. The 3′ end of the reporter includes a poly(A) site (PAS), the 3′ end sequence from MALAT1 (MALAT1), or the 3′ end sequence from H2AC18 (Histone) (see Methods for details). B , RT-qPCR analysis of random-primed cDNA following overexpression of each reporter. The reporter mRNA levels were normalized to the expression of a co-transfected renilla luciferase control plasmid. Then for each 3′ end pair, the mRNA level of the 5′ ss wild-type reporter was calculated relative to the 5′ ss mutant reporter. Statistical analysis from n = 3 independent samples was calculated using unpaired t-tests. C & D , RT-qPCR analysis of random-primed cDNA following overexpression of each reporter and dTAG-induced depletion of EXOSC3 ( C ) or ZFC3H1 ( D ). The reporter mRNA levels were normalized to the expression of a co-transfected firefly luciferase control plasmid. Then for each reporter, the mRNA level in the dTAG condition was calculated relative to the DMSO control condition. Statistical analysis from n = 3 independent samples was calculated using unpaired t-tests. E , Combined reporter RNA FISH and GAPDH immunofluorescence (IF) in wild-type HEK293T cells. The reporter RNA FISH signal is shown in green. GAPDH IF signal is shown in red. Nuclei were stained using DAPI and are depicted in blue. F , Boxplot displaying the quantification of the reporter RNA FISH signal in the nucleus versus cytoplasm in E (see Methods). For each 3′ end, the Nucleus/Cytoplasm FISH signal ratio of the 5′ wild-type reporter was normalized to the Nucleus/Cytoplasm FISH signal ratio of the 5′ ss mutant reporter. Y-axis values are formatted as log values. Statistical analysis from n = 10 (5′ ss- bGH , 5′ss Mut- bGH ), n = 12 (5′ss- MALAT1 3′ end), or n = 11 (5′ss Mut- MALAT1 3′ end) images per reporter was calculated using unpaired t-tests.

Article Snippet: Anti-FLAG mouse (Sigma-Aldrich, F1804-200UG) Anti-beta Tubulin (Invitrogen, PA1-16947) Anti-GAPDH (Santa Cruz, sc-365062) Anti-ZFC3H1 (CCDC131) (Bethyl, A301-456A) Anti-MTR4 (Abcam, AB70551) Anti-PABPN1 (Bethyl, A303-524A) Anti-ZCCHC8 (Bethyl, A301-806A) Anti-EXOSC3 (Proteintech, 15062-1-AP) Anti-U1-70K (Millipore, clone 964.1, 05-1588) Anti-U1A (Santa Cruz, sc-101149) Anti-U1C (Sigma, SAB4200188-200UL) Anti-ARS2 (Bethyl, A304-550A) Anti-CPSF100 (Bethyl, A301-581A) Anti-CPSF73 (Bethyl, A301-091A) Anti-CPSF30 (Bethyl, A301-585A) Anti-FLAG rabbit (Cell Signaling, 14793S) Anti-SC-35* (SRRM2) (Abcam, ab11826) *Recently, a study reported that anti-SC35, a widely used marker of nuclear speckles, recognizes SRRM2

Techniques: Sequencing, Mutagenesis, Quantitative RT-PCR, Random Primed, Over Expression, Expressing, Transfection, Luciferase, Control, Plasmid Preparation, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: A nuclear RNA degradation code for eukaryotic transcriptome surveillance

doi: 10.1101/2024.07.23.604837

Figure Lengend Snippet: A , Western blot analysis of EXOSC3 and GAPDH (loading control) levels in the EXOSC3 degron cell line that were mock-treated (DMSO) or dTAG-treated for 8 hours. B , Western blot analysis of ZFC3H1 and Tubulin (loading control) levels in the ZFC3H1 degron cell line that were mock-treated (DMSO) or dTAG-treated for 8 hours. C , Schematic of the U1 mutant snRNA expression approach used in D . Left: Wild-type (WT) U1 snRNP complexes will not recognize the 5′ ss Mut- bGH PAS reporter as the sequences are not complementary. Right: The exogenous mutant U1 snRNA will be loaded into U1 snRNP complexes, allowing U1 snRNP to recognize the bGH PAS-containing reporter with a Mut 5′ ss in its 3′ UTR. D , RT-qPCR of oligo d(T)-primed cDNA following overexpression of the reporter and the 5′ ss mutant-targeting U1 snRNA or a non-targeting U1 snRNA control. The reporter mRNA levels were first normalized to the expression of a co-transfected firefly luciferase control plasmid. The mRNA level of the reporter mRNA in the mutant-targeting U1 snRNA condition was then calculated relative to the non-targeting control U1 snRNA condition. Statistical analysis from n = 3 independent samples was calculated using an unpaired t-test. E , Representative images of reporter RNA FISH and SRRM2 immunofluorescence (IF) in wild-type HEK293T cells to monitor co-localization of the reporter RNA molecules and nuclear speckles. SRRM2 is used as a marker of nuclear speckles. The reporter RNA FISH signal is shown in green. SRRM2 IF signal is shown in red. Nuclei were stained using DAPI and are depicted in blue. F , Western blot analysis of FLAG-eGFP and Tubulin (loading control) levels after overexpressing the eGFP reporters with the bGH PAS or the MALAT1 3′ end in wild-type HEK293T cells for 48 hours. Proteins were detected using IR dye-conjugated secondary antibodies. G , Quantification of F using ImageQuant TL software. Each reporter protein level sample was first normalized to the loading control protein level. For each 3′ end pair, the relative protein levels were then calculated by dividing the reporter protein levels by one replicate of the 5′ ss mutant reporter. Statistical analysis of n = 3 independent samples was performed using unpaired t-tests. H , Western blot analysis of FLAG-eGFP and Tubulin (loading control) levels after overexpressing the eGFP reporters with the L3 PAS or the H2AC18 (Histone) 3′ end in wild-type HEK293T cells for 48 hours. Three independent samples are shown for each reporter.

Article Snippet: Anti-FLAG mouse (Sigma-Aldrich, F1804-200UG) Anti-beta Tubulin (Invitrogen, PA1-16947) Anti-GAPDH (Santa Cruz, sc-365062) Anti-ZFC3H1 (CCDC131) (Bethyl, A301-456A) Anti-MTR4 (Abcam, AB70551) Anti-PABPN1 (Bethyl, A303-524A) Anti-ZCCHC8 (Bethyl, A301-806A) Anti-EXOSC3 (Proteintech, 15062-1-AP) Anti-U1-70K (Millipore, clone 964.1, 05-1588) Anti-U1A (Santa Cruz, sc-101149) Anti-U1C (Sigma, SAB4200188-200UL) Anti-ARS2 (Bethyl, A304-550A) Anti-CPSF100 (Bethyl, A301-581A) Anti-CPSF73 (Bethyl, A301-091A) Anti-CPSF30 (Bethyl, A301-585A) Anti-FLAG rabbit (Cell Signaling, 14793S) Anti-SC-35* (SRRM2) (Abcam, ab11826) *Recently, a study reported that anti-SC35, a widely used marker of nuclear speckles, recognizes SRRM2

Techniques: Western Blot, Control, Mutagenesis, Expressing, Quantitative RT-PCR, Over Expression, Transfection, Luciferase, Plasmid Preparation, Immunofluorescence, Marker, Staining, Software

Journal: bioRxiv

Article Title: A nuclear RNA degradation code for eukaryotic transcriptome surveillance

doi: 10.1101/2024.07.23.604837

Figure Lengend Snippet: A , Schematic of the predicted domains within full-length ZFC3H1 and the expression constructs used to perform the FLAG-Immunoprecipitation experiments analyzed in B & C . Relevant residues are indicated as numbers above full-length ZFC3H1. The yellow rectangle represents the short acid-rich linear motif (SLiM) region. The mutated SLiM region within the N-term SLiM mutant (N-term ΔSLiM) construct is represented as a grey rectangle labeled Mut. The light blue rectangles depict coiled-coil (CC) regions. The light purple rectangle represents the zinc-finger domain (ZnF). The dark blue rectangle represents tetratricopeptide repeats (TPR). B & C , Western blotting analyses of proteins that co-immunoprecipitated with the indicated ZFC3H1 expression construct. The anti-FLAG western blots contain 1% input and 10% eluted IP sample. All other western blots contain 0.5% input and 30% eluted IP sample. D , Schematic depicting the interactions between ZFC3H1 and the associated proteins ARS2, U1 snRNP components (U1), subunits of CPSF (CPSF), PABPN1, and MTR4.

Article Snippet: Anti-FLAG mouse (Sigma-Aldrich, F1804-200UG) Anti-beta Tubulin (Invitrogen, PA1-16947) Anti-GAPDH (Santa Cruz, sc-365062) Anti-ZFC3H1 (CCDC131) (Bethyl, A301-456A) Anti-MTR4 (Abcam, AB70551) Anti-PABPN1 (Bethyl, A303-524A) Anti-ZCCHC8 (Bethyl, A301-806A) Anti-EXOSC3 (Proteintech, 15062-1-AP) Anti-U1-70K (Millipore, clone 964.1, 05-1588) Anti-U1A (Santa Cruz, sc-101149) Anti-U1C (Sigma, SAB4200188-200UL) Anti-ARS2 (Bethyl, A304-550A) Anti-CPSF100 (Bethyl, A301-581A) Anti-CPSF73 (Bethyl, A301-091A) Anti-CPSF30 (Bethyl, A301-585A) Anti-FLAG rabbit (Cell Signaling, 14793S) Anti-SC-35* (SRRM2) (Abcam, ab11826) *Recently, a study reported that anti-SC35, a widely used marker of nuclear speckles, recognizes SRRM2

Techniques: Expressing, Construct, Immunoprecipitation, Mutagenesis, Labeling, Western Blot

Journal: bioRxiv

Article Title: A nuclear RNA degradation code for eukaryotic transcriptome surveillance

doi: 10.1101/2024.07.23.604837

Figure Lengend Snippet: A , PAS-seq data tracks for the gene NEAT1_1 following depletion of EXOSC3, MTR4, ZFC3H1, or PABPN1 by RNAi in wild-type HEK293T cells. Each peak represents reads mapped to the 3′ end of mRNAs. B & C , Relative Expression (CPM: counts per million) of NEAT1_1 as reported by differential isoform expression analysis of PAS-seq data from HEK293T cells depleted of EXOSC3 ( B ), MTR4, ZFC3H1, or PABPN1 ( C ) by RNAi. NEAT1_1 exhibited at least a 1.5-fold increase in expression in all samples (log 2 FC > 0.585, FDR ≤ 0.05).

Article Snippet: Anti-FLAG mouse (Sigma-Aldrich, F1804-200UG) Anti-beta Tubulin (Invitrogen, PA1-16947) Anti-GAPDH (Santa Cruz, sc-365062) Anti-ZFC3H1 (CCDC131) (Bethyl, A301-456A) Anti-MTR4 (Abcam, AB70551) Anti-PABPN1 (Bethyl, A303-524A) Anti-ZCCHC8 (Bethyl, A301-806A) Anti-EXOSC3 (Proteintech, 15062-1-AP) Anti-U1-70K (Millipore, clone 964.1, 05-1588) Anti-U1A (Santa Cruz, sc-101149) Anti-U1C (Sigma, SAB4200188-200UL) Anti-ARS2 (Bethyl, A304-550A) Anti-CPSF100 (Bethyl, A301-581A) Anti-CPSF73 (Bethyl, A301-091A) Anti-CPSF30 (Bethyl, A301-585A) Anti-FLAG rabbit (Cell Signaling, 14793S) Anti-SC-35* (SRRM2) (Abcam, ab11826) *Recently, a study reported that anti-SC35, a widely used marker of nuclear speckles, recognizes SRRM2

Techniques: Expressing

Journal: STAR Protocols

Article Title: Using the dCas9-KRAB system to repress gene expression in hiPSC-derived NGN2 neurons

doi: 10.1016/j.xpro.2021.100580

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Article Snippet: Alternatively, we can apply a conventional western blot using enhanced chemiluminescent (ECL, GE, #RPN2236).

Techniques: Recombinant, Purification, Titration, Lysis, Western Blot, Plasmid Preparation, SYBR Green Assay, Bradford Protein Assay, RNA Sequencing Assay, Expressing, Software, CRISPR, Imaging, Microscopy, Real-time Polymerase Chain Reaction

Journal: Journal of NeuroEngineering and Rehabilitation

Article Title: Simulating space walking: a systematic review on anti-gravity technology in neurorehabilitation

doi: 10.1186/s12984-024-01449-z

Figure Lengend Snippet: Description of anti-gravity treadmill training parameters and settings

Article Snippet: Rose et al. [ ] , PD , Alter-G (AlterG Sports, AlterG Inc., California, USA) , NA , Force and electromyographic signals , Increased body weight support normalized extensor muscle activation abnormalities in PD patients..

Techniques: Control, Blocking Assay

Journal: Journal of NeuroEngineering and Rehabilitation

Article Title: Simulating space walking: a systematic review on anti-gravity technology in neurorehabilitation

doi: 10.1186/s12984-024-01449-z

Figure Lengend Snippet: Description of the reported interventions, outcomes and major findings

Article Snippet: Rose et al. [ ] , PD , Alter-G (AlterG Sports, AlterG Inc., California, USA) , NA , Force and electromyographic signals , Increased body weight support normalized extensor muscle activation abnormalities in PD patients..

Techniques: Activation Assay, Control, Functional Assay