asisi  (New England Biolabs)

Journal: bioRxiv

Article Title: Characterization of variants associated with Cerebral Small Vessel Disease identifies a functional SNV in Versican

doi: 10.64898/2026.03.16.712010

Figure Lengend Snippet: A) Schematic of the MPRA synthetic oligonucleotide including a forward primer (F), 102 bp of flanking sequence before, and 102 bp of flanking sequence after, the variant followed by a miniPromotor (miniP)- GFP construct, a 10 bp barcode and reverse primer (R). The oligo library was cloned into the pMPRAv3:Δluc:ΔxbaI vector and transfected into HEK293 cells. DNA and RNA was isolated separately and sequenced using MiSeq. B) Bioinformatic workflow for the MPRA analysis showing how allele-specific enhancers were identified. C) Fold change in expression for alelle specific enhancers in the indicated genes. Blue bars show significant allele-specific activity.

Article Snippet: The MPRA library incorporating GFP was created using 2μg of MiniGFP amplicon and 1μg of AsiSI linearized MPRA oligo library and assembled using NEBuilder HiFi DNA Assembly at 50C for 90 min, then purified with NucleoSpin PCR clean-up column, eluted and digested to remove remaining uncut vector by incubation with 25U AsiSI, and 5U RecBCD Plasmidsafe nuclease (NEB), 10μg BSA, 1 mM ATP in a 100μl reaction at 37 C overnight followed by NucleoSpin PCR clean-up column.

Techniques: Sequencing, Variant Assay, Construct, Clone Assay, Plasmid Preparation, Transfection, Isolation, Expressing, Activity Assay

Journal: bioRxiv

Article Title: Characterization of variants associated with Cerebral Small Vessel Disease identifies a functional SNV in Versican

doi: 10.64898/2026.03.16.712010

Figure Lengend Snippet: A) Intergenic region between FOXQ1 and FOXF2 showing location of variants that are significant allele-specific enhancers from the MPRA. B) Table showing the log fold change and adjusted p value for the expression difference between major and minor alleles (allele-specific enhancers).

Article Snippet: The MPRA library incorporating GFP was created using 2μg of MiniGFP amplicon and 1μg of AsiSI linearized MPRA oligo library and assembled using NEBuilder HiFi DNA Assembly at 50C for 90 min, then purified with NucleoSpin PCR clean-up column, eluted and digested to remove remaining uncut vector by incubation with 25U AsiSI, and 5U RecBCD Plasmidsafe nuclease (NEB), 10μg BSA, 1 mM ATP in a 100μl reaction at 37 C overnight followed by NucleoSpin PCR clean-up column.

Techniques: Expressing

Journal: bioRxiv

Article Title: Characterization of variants associated with Cerebral Small Vessel Disease identifies a functional SNV in Versican

doi: 10.64898/2026.03.16.712010

Figure Lengend Snippet: A) Schematic of where a 2 kb intronic enhancer region was deleted using two CRISPRs. B) Sequences of independent clones with the 2 kb region deleted. C) qPCR of endogenous VCAN in the deleted clones showing that VCAN V3 is significantly downregulated when the enhancer is deleted but there are no significant changes in V0, V1 and V2. D) Schematic of constructs with the major and minor alleles of rs13176921 in a 300bp oligo. E) Luciferase activity is decreased when the minor allele is present, relative to the major allele. N=3 biological replicates, with 3 technical replicates. F) Position weight matrix for NKX3.1 from JASPAR showing location of rs13176921 SNV. G) ChIP for NKX3.1 in the region of rs13176921, a positive control genomic region known to bind NKX3.1 (Chr 19) and a negative control region (Sat2). Statistics used a One Way ANOVA with Dunnett’s multiple comparison test, or the Student’s t-test.

Article Snippet: The MPRA library incorporating GFP was created using 2μg of MiniGFP amplicon and 1μg of AsiSI linearized MPRA oligo library and assembled using NEBuilder HiFi DNA Assembly at 50C for 90 min, then purified with NucleoSpin PCR clean-up column, eluted and digested to remove remaining uncut vector by incubation with 25U AsiSI, and 5U RecBCD Plasmidsafe nuclease (NEB), 10μg BSA, 1 mM ATP in a 100μl reaction at 37 C overnight followed by NucleoSpin PCR clean-up column.

Techniques: Clone Assay, Construct, Luciferase, Activity Assay, Positive Control, Negative Control, Comparison