Journal: bioRxiv

Article Title: KAS-CUT&Tag for direct mapping of transcription bubbles

doi: 10.64898/2026.05.15.725569

Figure Lengend Snippet: a , Schematic of the KAS-CUT&Tag workflow (created with BioRender.com). N 3 -kethoxal (magenta star) labels ssDNA, while a primary antibody (blue Y-shape) binds the target protein (blue oval). A secondary antibody (blue Y-shape) enhances recruitment of the pAG-Tn5 transposome (green), which is activated by Mg²⁺ to insert adapters (magenta lines) at protein-bound sites. After DNA purification, adapter-ligated fragments are biotinylated via click chemistry. Biotinylated DNA is either amplified by PCR to generate the input CUT&Tag library, or immunoprecipitated with streptavidin beads before PCR to construct the KAS-CUT&Tag library. b, Coverage-normalized Pol II-Ser5P CUT&Tag (CnT) signals (±N 3 -kethoxal), shown alongside KAS-seq data from Wu et al in K562 cells . Input CUT&Tag library corresponds to N 3 -kethoxal-treated CUT&Tag (CnT). c, d, Heatmaps (bottom) and average plots (top) centered on the TSSs of 12,397 protein-coding genes, showing Pol II-Ser5P CnT (±N 3 -kethoxal) ( c ) and KAS-seq data in K562 ( d ). Each row represents one gene. e, f, V-plots showing fragment size versus distance from TSSs for Pol II-Ser5P CnT (±N 3 -kethoxal) ( e ) and ATAC-seq (±N 3 -kethoxal) ( f ). ATAC-seq (–N 3 -kethoxal) is from Tak et al , while ATAC-seq (+N 3 -kethoxal) is from Kim et al and Lyu et al . g, Density plot of Tn5 fragment ends within a 400 bp window centered on TSSs.

Article Snippet: After washing by wash buffer, beads were resuspended in 300-wash buffer (wash buffer + 50 mM NaCl) containing pAG-Tn5 transposase (1:10 dilution; EpiCypher, 15-1117) and incubated at 4 °C for 1 h. Excess pAG-Tn5 was removed by washing with 300-wash buffer, and tagmentation was performed by incubating in CUTAC-DMF tagmentation buffer (20% N,N-dimethylformamide (Sigma-Aldrich, D8654), 10 mM TAPS PH8.5, 5 mM, 0.05% Triton X-100, 5 mM MgCl 2 ) at 37 °C for 1 h. Following tagmentation, beads were washed with TAPS buffer (10 mM TAPS, 0.2 mM EDTA) and resuspended in release solution (5 μl total; 10 mM TAPS, 0.1% SDS, and 1:10 Thermolabile Proteinase K(New England Biolabs, P8111S).

Techniques: DNA Purification, Amplification, Immunoprecipitation, Construct

Journal:

Article Title:

doi:

Figure Lengend Snippet: ( a ) CRISPR-sciATAC workflow with initial barcoding, nuclei pooling and re-splitting, and then second round barcoding. ( b ) Comparison of bulk ATAC-seq chromatin accessibility profiles from K562 cells using Tn5 and TnY transposases and aggregated CRISPR-sciATAC single cell profiles from 11,104 cells. ( c ) Guide RNA (gRNA) reads mapping to human or mouse CRISPR libraries ( n = 1986 cells). ( d ) ATAC reads mapping to human or mouse genomes ( n = 721 cells). For display purposes, we removed one cell that had >10-fold the average number of ATAC reads. ( e ) Concordance between the percent of ATAC and gRNA reads mapping to the human and mouse genomes and human and mouse gRNA libraries, respectively, for each cell ( n = 496 cells). ( f ) ATAC-seq fragment size distribution from K562 cells of bulk ATAC-seq data, aggregated CRISPR-sciATAC single cell profiles from 11,104 cells and one representative single cell from CRISPR-sciATAC. ( g ) Number of CRISPR gRNAs detected per cell. ( h ) Proportion of cells with 1, 2, or more than 2 gRNAs.

Article Snippet: The identified ViPar transposase was synthesized (Twist Bioscience) and cloned into the vector pTXB1 (NEB, N6707S).

Techniques: CRISPR, Single Cell, Sequencing, Comparison

Journal: STAR Protocols

Article Title: Protocol to identify SINE-VNTR-Alu regulators using genome-wide screening in human K562 cells

doi: 10.1016/j.xpro.2026.104468

Figure Lengend Snippet: Illustration of the PiggyBac system for exogenous DNA insertion into the human genome After the co-transfection of the PiggyBac transposase expression plasmid and PiggyBac cloning and expression vector, the expressed PiggyBac transposase cut the Ins, Insulator. ITR, inverted terminal repeat. Adapted from the website of System Biosciences.

Article Snippet: Obtain PiggyBac transposase expression plasmid from System Biosciences (Cat# PB210PA-1).

Techniques: Cotransfection, Expressing, Plasmid Preparation, Cloning

Journal: STAR Protocols

Article Title: Protocol to identify SINE-VNTR-Alu regulators using genome-wide screening in human K562 cells

doi: 10.1016/j.xpro.2026.104468

Figure Lengend Snippet: The map of PiggyBac cloning and expression plasmid The schematic illustrates a plasmid backbone containing an ampicillin resistance marker, a multiple cloning site (MCS) for insertion of the desired expression cassette, and an SV40 polyadenylation signal to facilitate proper transcriptional termination.

Article Snippet: Obtain PiggyBac transposase expression plasmid from System Biosciences (Cat# PB210PA-1).

Techniques: Cloning, Expressing, Plasmid Preparation, Marker

Journal: STAR Protocols

Article Title: Protocol to identify SINE-VNTR-Alu regulators using genome-wide screening in human K562 cells

doi: 10.1016/j.xpro.2026.104468

Figure Lengend Snippet: FACS analysis showed the GFP+ cell percentage after plasmid transfection The transfection of PiggyBac-SVA-GFP-pA and PiggyBac-SVA-AAA-GFP-pA into HEK293T cells can produce a GFP+ cell population, while the transfection of PiggyBac-SVA-A-GFP-pA and PiggyBac-SVA-AA-GFP-pA cannot, indicating the frame shift of translation.

Article Snippet: Obtain PiggyBac transposase expression plasmid from System Biosciences (Cat# PB210PA-1).

Techniques: Plasmid Preparation, Transfection

Journal: STAR Protocols

Article Title: Protocol to identify SINE-VNTR-Alu regulators using genome-wide screening in human K562 cells

doi: 10.1016/j.xpro.2026.104468

Figure Lengend Snippet: Illustration of the PiggyBac system for exogenous DNA insertion into the human genome After the co-transfection of the PiggyBac transposase expression plasmid and PiggyBac cloning and expression vector, the expressed PiggyBac transposase cut the Ins, Insulator. ITR, inverted terminal repeat. Adapted from the website of System Biosciences.

Article Snippet: Plasmid: PiggyBac transposase , System Biosciences , Cat# PB210PA-1.

Techniques: Cotransfection, Expressing, Plasmid Preparation, Cloning

Journal: STAR Protocols

Article Title: Protocol to identify SINE-VNTR-Alu regulators using genome-wide screening in human K562 cells

doi: 10.1016/j.xpro.2026.104468

Figure Lengend Snippet: The map of PiggyBac cloning and expression plasmid The schematic illustrates a plasmid backbone containing an ampicillin resistance marker, a multiple cloning site (MCS) for insertion of the desired expression cassette, and an SV40 polyadenylation signal to facilitate proper transcriptional termination.

Article Snippet: Plasmid: PiggyBac transposase , System Biosciences , Cat# PB210PA-1.

Techniques: Cloning, Expressing, Plasmid Preparation, Marker

Journal: STAR Protocols

Article Title: Protocol to identify SINE-VNTR-Alu regulators using genome-wide screening in human K562 cells

doi: 10.1016/j.xpro.2026.104468

Figure Lengend Snippet: FACS analysis showed the GFP+ cell percentage after plasmid transfection The transfection of PiggyBac-SVA-GFP-pA and PiggyBac-SVA-AAA-GFP-pA into HEK293T cells can produce a GFP+ cell population, while the transfection of PiggyBac-SVA-A-GFP-pA and PiggyBac-SVA-AA-GFP-pA cannot, indicating the frame shift of translation.

Article Snippet: Plasmid: PiggyBac transposase , System Biosciences , Cat# PB210PA-1.

Techniques: Plasmid Preparation, Transfection