length plasmid sequencing  (Eurofins)

Journal: bioRxiv

Article Title: Role of a childhood cancer-linked BRIP1/FANCJ germline variant in genomic instability and cancer cell vulnerability

doi: 10.64898/2026.03.24.714005

Figure Lengend Snippet: a, Three-generation pedigree of an 11-year-old pediatric patient diagnosed with cancer investigated in this study. b, Whole-exome sequencing (WES) of peripheral-blood-derived DNA from the patient and both parents identified a novel missense variant in BRIP1 (c.485G>A, p.Arg162Gln) located in the nuclear localization signal (NLS), inherited from the mother, and a missense variant in HIPK2 (c.1804A>C, p.Thr602Pro), inherited from the father. c, Coomassie-stained SDS-PAGE of purified recombinant BRIP1/FANCJ WT and BRIP/FANCJ R162Q proteins. d, FAM-labeled 5’ overhang DNA substrate unwinding assay using increasing concentrations of BRIP1 WT or R162Q (1-20 nM) as indicated. Upper signal indicates the duplex substrate, the lower one the unwound FAM-labelled oligonucleotide. A representative assay is shown e, Quantification of DNA unwinding assays (n=3) shown as percentage of unwound substrate (mean ± SEM).

Article Snippet: The correct sequence of pFB-FANCJ-STREP-WT and pFB-FANCJ-R162Q-STREP constructs were verified by full length plasmid sequencing by Eurofins.

Techniques: Sequencing, Derivative Assay, Variant Assay, Staining, SDS Page, Purification, Recombinant, Labeling

Journal: bioRxiv

Article Title: Role of a childhood cancer-linked BRIP1/FANCJ germline variant in genomic instability and cancer cell vulnerability

doi: 10.64898/2026.03.24.714005

Figure Lengend Snippet: a, Schematic overview of the generation of HCT116 cell pools stably overexpressing BRIP1 WT or BRIP1 R162Q. BRIP1 WT or R162Q constructs contained C-terminal c-Myc and HA epitope tags. b, Representative Sanger sequencing traces confirming the presence of BRIP1 WT or BRIP1 R162Q in transduced HCT116 cells. c, d, Immunoblot analysis confirming expression of BRIP1 and Myc-tagged proteins in HCT116 cells expressing BRIP1 WT or R162Q, detected using BRIP1 and Myc antibodies. Vinculin served as a loading control. e, j, Functional characterization of HCT116 cells expressing BRIP1 WT or R162Q by colony formation assays following e, mitomycin C (MMC) (0.5;1; 2; 3; 4 μM/ml), g, ionizing radiation (IR) (2; 3; 4; 5 Gy), and j, hydroxyurea (HU) treatment 50;100;150;200;250 μM/ml). Representative colony plates (upper panels) and quantification of survival fractions (lower panels) are shown. Quantification was performed using EagleEye analysis software. Data represent the mean ± SD of three independent biological replicates.

Article Snippet: The correct sequence of pFB-FANCJ-STREP-WT and pFB-FANCJ-R162Q-STREP constructs were verified by full length plasmid sequencing by Eurofins.

Techniques: Stable Transfection, Construct, Sequencing, Western Blot, Expressing, Control, Functional Assay, Software

Journal: bioRxiv

Article Title: Decoding the MYC locus reveals a druggable ultraconserved RNA element

doi: 10.64898/2026.01.29.702547

Figure Lengend Snippet: A . Base-pair phenotype scores of the 3’ UTR and zoom-in to the sg723 target site. Blue and red stars highlight 3’ UTR base pairs with the strongest positive and negative phenotype scores, respectively. Amplicon sequencing reads indicating indels were also shown (only those with frequency > 0.4%). B . Competitive growth assay comparing HF-SpRY-Cas9 D11 cells expressing sg723 or a non-targeting control sgRNA. Data were normalized to Day 0. C . Relative cell viability (WST-1) comparing two clonal cell lines deleting the ultraconserved element to a cell line targeted by a control sgRNA. Data were normalized to WT. D . Relative cell viability (WST-1) comparing cells overexpressing GFP alone, GFP fused to a full-length MYC gene (MYC-WT, MYC-𝛥8, or MYC-𝛥2) using plasmids. Data were normalized to GFP vector. E . Four ASOs targeting the ultraconserved element. F - J . Relative cell viability (WST-1) in indicated cell lines transfected with a control ASO or one of four ASOs targeting the ultraconserved RNA element. Data were normalized to ASO-ctrl. K . MYC mRNA knockdown with siRNAs in HEK293T cells. Data were normalized to siCtrl. L . MYC knockdown did not significantly reduce HEK293T cell viability. Data were normalized to siCtrl. ns, not significant, *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001.

Article Snippet: Final construct verification was conducted through full-length plasmid sequencing performed by Plasmidsaurus to confirm each plasmid sequence.

Techniques: Amplification, Sequencing, Growth Assay, Expressing, Control, Plasmid Preparation, Transfection, Knockdown

Journal: bioRxiv

Article Title: Decoding the MYC locus reveals a druggable ultraconserved RNA element

doi: 10.64898/2026.01.29.702547

Figure Lengend Snippet: A . HA-tagged MYC was expressed from a plasmid containing the full-length MYC gene, including introns and UTRs. The HA-MYC-𝛥8 and -𝛥2 variants delete 8-nt and 2-nt from the element, respectively. Plasmids were transiently transfected into U87MG cells for 48 h, and MYC localization was assessed by HA-tag immunofluorescence staining. B . Representative immunofluorescence images. C . Quantification of the nucleus/cytoplasm ratio as in . MYC-WT: N=27; MYC-𝛥8: N=35; MYC-𝛥2: N=55. ***: p<0.001, ****: p<0.0001.

Article Snippet: Final construct verification was conducted through full-length plasmid sequencing performed by Plasmidsaurus to confirm each plasmid sequence.

Techniques: Plasmid Preparation, Transfection, Immunofluorescence, Staining

Journal: Reproduction (Cambridge, England)

Article Title: mTORC1-dependent suppression of autophagic activity in somatic cell nuclear transfer mouse embryos

doi: 10.1530/REP-25-0338

Figure Lengend Snippet: Preimplantation autophagic activity compared in IVF and SCNT embryos. (A) Representative fluorescent images of IVF and SCNT embryos at various times after fertilization or activation. From left to right, 24, 48, 72, and 96 h later were shown, IVF embryos on the upper row, and SCNT embryos on the lower row. GFP-LC3 (green), RFP-LC3ΔG (red). (B) Preimplantation autophagic activity in IVF and SCNT embryos. The x -axis shows the elapsed time, and the y -axis shows the relative RFP/GFP values for each embryo based on the 24-h time point. IVF (blue), SCNT (orange), lighter-colored areas indicate SEM. Two-tailed t -tests were performed at 36, 48, 60, and 72 h. IVF n = 35, SCNT n = 6. (C) Autophagic activity in IVF and SCNT embryos that were treated to enhance gene expression from 24 to 72 h after insemination or activation. The x -axis shows the elapsed time, and the y -axis shows the relative RFP/GFP values for each embryo based on the 24-h time point. IVF (blue), Kdm4d-overexpressed (red), TSA-treated (green), lighter-colored areas indicate SEM. IVF: n = 12, Kdm4d: n = 38, TSA: n = 18. (D) Autophagic activity up to 48 h after activation in SCNT embryos treated to enhance gene expression. Kdm4d-overexpressed (red), TSA-treated (green), lighter-colored areas indicate SEM. The broken lines indicate embryos that were subjected to the respective treatments, but embryonic development was arrested after the 4-cell stage. Two-tailed t -tests were performed at 48 h for each treatment group. Kdm4d arrest n = 22, TSA arrest n = 35.

Article Snippet: Briefly, the pcDNA3.1 plasmid carrying the full-length mouse Kdm4d sequence (61553, Addgene, USA) was linearized by XbaI, and it was used as a template for in vitro transcription using mMESSAGE mMACHINE T7 ULTRA Transcription kits (AM1345, Thermo Fisher Scientific).

Techniques: Activity Assay, Activation Assay, Two Tailed Test, Gene Expression