kpni hf restriction enzymes  (New England Biolabs)

Journal: iScience

Article Title: Impact of Tau overexpression on DNA replication dynamics in centromeres of human neural progenitor cells

doi: 10.1016/j.isci.2026.114707

Figure Lengend Snippet: Validation of the SMARD molecules of the ChrY and Chr21 centromeric regions (A) Representative SMARD molecule shows different steps of DNA replication including initiation (ORIs) (◆), fork progression from IdU (red) to CldU (green) (►: 5′-3’; ◄: 3′-5′), and termination (►◄). Replication initiation events are predicted to be at the centers of the red signals, which are flanked by two green signals. Below the four SMARD molecules, there are two diagrams, which summarize the replication processes captured by the SMARD. The top diagram summarizes the fork progressions: two forks move from 5′ to 3’ (the vertical lines pointing up), while one fork moves from 3′ to 5’ (the vertical line pointing down). The bottom diagram summarizes the fork initiation event (ORI). (B) A diagram of the ChrY centromeric SMARD molecule. ChrY (57 Mbp) was cut by the KpnI restriction enzyme to generate a 570-kbp DNA segment of ChrY SMARD molecule (the black line), which is composed of a 384-kbp DNA region of ChrY α-satellite DNA (the blue line) and a 186-kbp DNA region of flanking DNA (the red line). A representative ChrY SMARD molecule is also shown. Blue, signals detected using a FISH probe for ChrY α-satellite; red, IdU incorporation; green, CldU incorporation. (C) Long-read DNA sequencing confirmed the size and molecular composition of the ChrY molecule detected by SMARD. The gray color indicates the α-satellite HOR of ChrY (Y-HOR, 353 kbp). The insert to the right shows the other repeat elements flanking the Y-HOR in different colors. The heatmap visualized by StainedGlass indicates the pairwise sequence identity with the percentage identity of 5-kbp fragments. (D) A diagram of the Chr21 centromeric SMARD molecule. Chr21 (37 Mbp) was cut by the BamHI restriction enzyme to generate a 1.06-Mbp DNA segment of the hapl.2 (haplotype 2) Chr21 SMARD molecule (the black line), which is composed of an 846-kbp DNA region of Chr21 α-satellite DNA (the blue line) and a 214-kbp DNA region of flanking DNA (the red line). A representative Chr21 SMARD molecule is also shown. Blue, signals detected using a FISH probe for Chr21 α-satellite; red, IdU incorporation; green, CldU incorporation. (E) Long-read DNA sequencing confirms the size and molecular composition of the hapl.2 Chr21 SMARD molecule. The gray color indicates the α-satellite HOR of Chr21 (21-HOR, 1.18 Mbp). The insert to the right shows the other repeats flanking the 21-HOR haplotype 2 in different colors. The heatmap visualized by StainedGlass indicates the pairwise sequence identity with the percentage identity of 5-kbp fragments.

Article Snippet: The ChrY SMARD segment is obtained by digesting the genomic DNA with the restriction enzyme KpnI at GGTACˆC sequence (NEB, R3142).

Techniques: Biomarker Discovery, DNA Sequencing, Sequencing

Journal: iScience

Article Title: Impact of Tau overexpression on DNA replication dynamics in centromeres of human neural progenitor cells

doi: 10.1016/j.isci.2026.114707

Figure Lengend Snippet: Pausing events at Y α-satellites and secondary ORI activations under Tau overexpression (A) Representative analysis of the aligned centromeric ChrY SMARD molecules from the CIMH-NP-3-DOX cells ( N = 36). The region detected by biotin-labeled, Y-specific α-satellite DNA FISH probe is shown in blue, and the flanking sequence is shown in red. On the top, two diagrams indicate the fork locations and initiation events of the SMARD molecules. The 570-kbp SMARD molecules are aligned to the yellow lines that correspond to the external KpnI cut sites and to the transition region between Y α-satellite and flanking regions (middle yellow line). SMARD molecules are arranged in the following order: initiation events (ORIs), fork progressing, and termination events. The initiation events are marked by orange diamonds (◆); the orientation of the replication forks is shown as the transition from IdU (red) to CldU (green), represented by yellow arrows ► 5′-3′ or ◄ 3′-5′; and the termination events (►◄). A 30-kbp scale bar is illustrated at the bottom of the aligned SMARD molecules; 1 kbp is 0.38 μm. (B and C) Quantification of the replication forks and ORIs within every 30 kbp (bin) of the SMARD molecules from several replicates for CIMH-NP-3-DOX (n = 36), wt Tau (n = 34,35), and php −/+DOX (n = 31,34). n = number of molecules. Graphs were generated using the same CIMH-NP-3 −DOX dataset. The Z -score was calculated as z = (x - μ)/σ, where x is the observed value, μ is the mean, and σ is the standard deviation (one-tailed). Values ≥ 1.645 were considered significant∗. Clone #6 for wt Tau and clone #6 for php Tau were used for the SMARD analysis.

Article Snippet: The ChrY SMARD segment is obtained by digesting the genomic DNA with the restriction enzyme KpnI at GGTACˆC sequence (NEB, R3142).

Techniques: Over Expression, Labeling, Sequencing, Generated, Standard Deviation, One-tailed Test

Journal: iScience

Article Title: Impact of Tau overexpression on DNA replication dynamics in centromeres of human neural progenitor cells

doi: 10.1016/j.isci.2026.114707

Figure Lengend Snippet: Tau overexpression alters global replication initiation and reduces origin initiation frequency in α-satellites of ChrY and Chr21 (A) A schematic of the preparation of NS-seq. Proliferating cells are used to extract the genomic DNA and the newly synthetized strands of DNA. The newly synthesized strands of DNA are isolated from genomic DNA by sucrose gradient (ranging size 0.5 to 2 kbp, not including the lagging strand Okazaki fragments). After the digestion of DNA without RNA primers at the 5′ by λ exonuclease to select only the RNA-primed DNA, the sequencing library is prepared to sequence and align to a reference genome (T2T_v2 or CIMH-NP-3 parental cell line). Created in BioRender. Balzano, E. (2025) https://BioRender.com/zx7j271 . (B) Genome browser snapshots showing the ORIs mapped by NS-seq in 773 kbp of the q-arm of Chr21, located at 21q21 (aligned using T2T_v.2). Each peak corresponds to an active ORI. Gray arrowheads mark origins active in all three conditions (CIMH-NP-3 parental, wt Tau + DOX, and php Tau + DOX cells). Blue and red arrowheads mark origins active or inactive only in wt/php Tau, respectively. (C) Venn diagram comparing ORI locations in doxycycline-treated CIMH-NP-3 parental, wt Tau, and php Tau cells. Cells overexpressing either wt Tau or php Tau showed fewer origins, with 30% of same origins shared under all conditions. (D) Bar plot displaying the number of active ORIs in doxycycline-treated and untreated CIMH-NP-3 parental and wt Tau or php Tau cells, showing a 20%–25% reduction in ORIs after wt Tau or php Tau overexpression. Unpaired t test was used for statistical significance; not significant (ns), ∗∗ p ≤ 0.001; n = 2 independent experiments; error bars represent the standard deviation (SD). (E and F) Genome browser snapshots showing ORIs mapped on the centromeric regions (α-satellite and flanking regions used in the SMARD assay) of ChrY (E) and Chr21 (F). NS-seq reads were aligned to a custom reference generated from PacBio long-read sequencing of CIMH-NP-3 ChrY (KpnI-digested) and Chr21 (BamHI-digested, haplotype 2). At the top of each chromosome ideogram, the blue lines mark the in silico probe location of Y α-satellites using DYZ3 Y-A and B primers and the 21 α-satellites using D13/21 ZL-3A and 1B primers. The right side shows a zoomed-in view of a single origin within the centromeric region. (G) Violin plot of initiation strength expressed in log 2 of read counts, measured by normalized reads per origin to genomic controls shown in (E) and (F). Statistical analysis was calculated using Wilcoxon signed-rank test; not significant (ns), ∗∗∗∗ p ≤ 0.0001; n = 2 independent experiments. ORIs, origins of replication; NS-seq, nascent strand sequencing.

Article Snippet: The ChrY SMARD segment is obtained by digesting the genomic DNA with the restriction enzyme KpnI at GGTACˆC sequence (NEB, R3142).

Techniques: Over Expression, Synthesized, Isolation, Sequencing, Standard Deviation, Generated, In Silico