recombinant hbd-2 (300 - 49)  (PeproTech)

Journal: Science Advances

Article Title: Genomic profiling of native R loops with a DNA-RNA hybrid recognition sensor

doi: 10.1126/sciadv.abe3516

Figure Lengend Snippet: ( A ) Schematic depiction of the domain structure of RNase H1 protein. The HBD domain of RNase H1 is responsible for the specific recognition of the DNA-RNA hybrids . GST-His 6 -HBD and GST-His 6 -2×HBD expression constructs are shown below. ( B ) Analysis of the purified GST-His 6 -HBD and GST-His 6 -2×HBD proteins by SDS–polyacrylamide gel electrophoresis (PAGE) and Coomassie blue staining. ( C to G ) EMSAs showing GST-His 6 -2×HBD prefers the DNA-RNA hybrid (C), compared to ssDNA (D), dsDNA (E), ssRNA (F), and dsRNA (G). Fluorescent probes (30 nM) were incubated with increasing concentrations of GST-His 6 -2×HBD (2×HBD) as the indicator for binding. The complexes were resolved with a 6% native polyacrylamide gel and were imaged with a Typhoon FLA-9500. GST-His 6 -2×HBD: DNA-RNA hybrid complexes are indicated by a bracket. ( H ) Biolayer interferometry assay of DNA-RNA hybrid and GST-His 6 -2×HBD. Biotinylated DNA-RNA hybrid was immobilized on streptavidin biosensors and incubated with a range of GST-His 6 -2×HBD (from 6.25 to 200 nM) to measure the response in an Octet Red96 instrument. ( I ) EMSAs analysis of GST-His 6 -2×HBD with probes of different GC contents.

Article Snippet: Two micrograms of recombinant GST-His 6 -2×HBD protein or S9.6 (anti–DNA-RNA hybrid antibody, clone S9.6; Millipore, MABE1095) was added to incubate with the bead-bound cells by rotating overnight at 4°C.

Techniques: Expressing, Construct, Purification, Polyacrylamide Gel Electrophoresis, Staining, Incubation, Binding Assay

Journal: Science Advances

Article Title: Genomic profiling of native R loops with a DNA-RNA hybrid recognition sensor

doi: 10.1126/sciadv.abe3516

Figure Lengend Snippet: ( A ) Schematic presentation of DRIPc-seq with GST-His 6 -2×HBD protein. ( B and C ) UCSC genome browser tracks of 2×HBD-DRIPc-seq, DRIPc-seq (GSE102474) , PRO-seq, and TT-seq reads density at the NCK2 , UXS1 (B), MRPS9 , and POU3F3 (C) loci. Read density was normalized by reads per million (r.p.m.). ( D ) Heatmap and metagene plots of 2×HBD-DRIPc-seq, the published DRIPc-seq, PRO-seq, and TT-seq signals in the plus and minus strands. ( E ) Scatter plot of the 2×HBD-DRIPc-seq counts and S9.6–DRIPc-seq counts with all of the protein-coding genes. The Pearson correlation coefficient is shown. ( F ) The genome-wide Pearson correlation heatmap of 2×HBD-DRIPc-seq, S9.6-DRIPc-seq, and TT-seq showing densities within all protein-coding genes.

Article Snippet: Two micrograms of recombinant GST-His 6 -2×HBD protein or S9.6 (anti–DNA-RNA hybrid antibody, clone S9.6; Millipore, MABE1095) was added to incubate with the bead-bound cells by rotating overnight at 4°C.

Techniques: Genome Wide

Journal: Science Advances

Article Title: Genomic profiling of native R loops with a DNA-RNA hybrid recognition sensor

doi: 10.1126/sciadv.abe3516

Figure Lengend Snippet: ( A ) Overview of the R loop CUT&Tag workflow. Cells were immobilized on concanavalin A (ConA)–coated magnetic beads, followed by cell permeabilization. GST-His 6 -2×HBD or S9.6 is used to recognize the R loops in the presence or absence of RNase A. Anti-GST, anti-HisTag, or secondary antibodies were applied to enhance the tethering of pA-Tn5 transposome at the GST-His 6 -2×HBD or S9.6-bound sites. After extensive wash, the pA-Tn5 transposome is activated to integrate the adapters on the chromatin. ( B ) CUT&Tag library preparation with Bst 2.0 WarmStart and Q5 polymerase. Strand displacement was performed with Bst 2.0, followed by library amplification with Q5 DNA polymerase. ( C ) Three different approaches for R loop CUT&Tag analysis. ( D ) LabChip analysis of R loop CUT&Tag library demonstrating the library size ranges from 220 to 700 bp with an average size of 405 bp. UM, upper marker; LM, lower marker. ( E ) Alignment rates of R loop CUT&Tag reads to the human hg38 and E. coli spiked-in genomes. RNase A treatment markedly decreases the alignment rates of CUT&Tag reads to the human genome, suggesting the specificity of GST-His 6 -2×HBD and S9.6 on R loop recognition. ( F ) UCSC genome browser tracks of CUT&Tag signals at the NPM1 and YY1AP1 loci. The tracks were normalized by reads per million, and the RNase A–treated groups were further normalized with the E. coli spike-in control.

Article Snippet: Two micrograms of recombinant GST-His 6 -2×HBD protein or S9.6 (anti–DNA-RNA hybrid antibody, clone S9.6; Millipore, MABE1095) was added to incubate with the bead-bound cells by rotating overnight at 4°C.

Techniques: Magnetic Beads, Library Amplification, Marker, Control

Journal: Science Advances

Article Title: Genomic profiling of native R loops with a DNA-RNA hybrid recognition sensor

doi: 10.1126/sciadv.abe3516

Figure Lengend Snippet: ( A ) Analysis of R loop CUT&Tag signals at all of the peaks from GST-His 6 -2×HBD and S9.6 CUT&Tag. RNase A digestion markedly reduced the CUT&Tag signals at those peaks, suggesting great specificity of GST-His 6 -2×HBD and S9.6 with R loops. ( B ) Heatmap profiles of CUT&Tag signals with or without RNase A treatment. ( C ) Annotation of CUT&Tag peaks showing the localization of the majority of R loops at the promoter regions. The genomic features are shown on the right. UTR, untranslated region. ( D ) Violin plot of CUT&Tag peak width with three different approaches. Wilcoxon test was used to test the statistical differences. CUT&Tag analysis with anti-HisTag antibody and GST-His 6 -2×HBD provides a superior resolution of R loop mapping. ( E and F ) Scatter plots of the log 2 fold changes of R loop signals detected by anti- HisTag with RNase A (+/−) versus the log 2 fold changes of anti-GST and RNase A (+/−) (E) or log 2 fold changes of S9.6 and RNase A (+/−) (F). CUT&Tag analysis with anti-HisTag antibody and GST-His 6 -2×HBD is the most specific approach for R loop mapping. ( G to I ) Scatter plots of CUT&Tag signals from three different approaches. Pearson correlation was performed, and the r values are shown.

Article Snippet: Two micrograms of recombinant GST-His 6 -2×HBD protein or S9.6 (anti–DNA-RNA hybrid antibody, clone S9.6; Millipore, MABE1095) was added to incubate with the bead-bound cells by rotating overnight at 4°C.

Techniques:

Journal: Science Advances

Article Title: Genomic profiling of native R loops with a DNA-RNA hybrid recognition sensor

doi: 10.1126/sciadv.abe3516

Figure Lengend Snippet: ( A to D ) UCSC genome browser tracks of CUT&Tag signals at the NPM1 , RPL13A , YY1AP1 , and FUS loci. The tracks were normalized by reads per million and the RNase H–treated groups were further normalized with the E. coli spike-in control. ( E ) Alignment rates of R loop CUT&Tag reads to the human hg38 and E. coli spiked-in genomes. Four-hour RNase H treatment markedly reduces the alignment rates of CUT&Tag reads to the human genome and increases the alignment rates of reads to E. coli spiked-in genomes. ( F and G ) Heatmap and metaplot analysis of R loop CUT&Tag signals at all of the peaks from GST-His 6 -2×HBD and S9.6 CUT&Tag. RNase H digestion markedly decreases the CUT&Tag signals at those peaks, demonstrating great specificity of GST-His 6 -2×HBD and S9.6 on R loop recognition. ( H to J ) Reproducibility of R loop CUT&Tag methods. Biological replicates were performed, and the Pearson correlation was calculated with the reads per million at R loop peaks.

Article Snippet: Two micrograms of recombinant GST-His 6 -2×HBD protein or S9.6 (anti–DNA-RNA hybrid antibody, clone S9.6; Millipore, MABE1095) was added to incubate with the bead-bound cells by rotating overnight at 4°C.

Techniques: Control

Journal: Science Advances

Article Title: Genomic profiling of native R loops with a DNA-RNA hybrid recognition sensor

doi: 10.1126/sciadv.abe3516

Figure Lengend Snippet: ( A and B ) Track examples of HEK293T PRO-seq, GST-His 6 -2×HBD CUT&Tag, S9.6 CUT&Tag, MapR , R-ChIP , DRIPc-seq , and TT-seq signals at the HSPD1 (A) and GRK6 (B) loci. The reads were aligned to the human hg38 genome, and the signals were normalized by reads per million. ( C ) PCA plot showing R loop CUT&Tag, MapR, and R-ChIP clustered together. ( D ) Fingerprint plots of R loop CUT&Tag, MapR, and R-ChIP. w.r.t., with respect to. ( E and F ) Metaplots of signals detected by different R loop mapping methods, PRO-seq, and TT-seq around the 2-kb window of the TSSs and TESs. Strand-specific signals from PRO-seq, TT-seq, DRIPc-seq, and R-ChIP were used for plotting. ( G ) Heatmap analysis of PRO-seq, TT-seq, and R loop mapping methods at the TSS of transcriptionally active genes (the reads per million of PRO-seq signals at TSS, >1; n = 13,220). The heatmaps are sorted by the GST-His 6 -2×HBD CUT&Tag signals. R loop CUT&Tag assays, MapR, and R-ChIP have enrichment at the TSS, while DRIPc-seq does not show this trend. ( H and I ) Scatter plots of R loop CUT&Tag and MapR reads per kilobase, per million mapped reads (RPKM) values (H) or R-ChIP RPKM values (I) at TSS. The r values were calculated by Pearson correlation.

Article Snippet: Two micrograms of recombinant GST-His 6 -2×HBD protein or S9.6 (anti–DNA-RNA hybrid antibody, clone S9.6; Millipore, MABE1095) was added to incubate with the bead-bound cells by rotating overnight at 4°C.

Techniques:

Journal: Science Advances

Article Title: Genomic profiling of native R loops with a DNA-RNA hybrid recognition sensor

doi: 10.1126/sciadv.abe3516

Figure Lengend Snippet: ( A ) Scheme of calculation of signals at TSS and gene body. The signals were normalized by RPKM. ( B ) Box plots of RPKM values at the TSS and gene body from 13,181 transcriptional active genes. ( C ) Scatter plot of R loop CUT&Tag signals at the TSS and gene body showing that R loop CUT&Tag is capable of genome wide detecting the R loop in the gene body. ( D and E ) Scatter plots of MapR (D) and R-ChIP (E) RPKM signals at the TSS and gene body. ( F ) Heatmap plots of the 3769 genes with R loop signals at gene body ( G and H ) The R loop signals at gene body were negatively correlated with gene lengths. ( I and J ) The gene body R loop signals positively correlate with PRO-seq (I) and H3K36me3 (J) signals at the gene body. ( K ) Gene ontology (GO) analysis of the 3769 genes indicates that R loop may be involved in the regulation of various key biological processes. ( L ) Track examples of HEK293T PRO-seq, GST-His 6 -2×HBD CUT&Tag, and S9.6 CUT&Tag signals at the YY1 and ZNF557 genomic loci. The reads were normalized by reads per million, and the enhancers are indicated. ( M ) Heatmap analysis of R loop CUT&Tag signals at 3830 intergenic regions. The heatmaps were sorted by the GST-His 6 -2×HBD CUT&Tag signals, and the H3K27ac signals in HEK293T are shown. H3K27ac, histone 3 lysine 27 acetylation.

Article Snippet: Two micrograms of recombinant GST-His 6 -2×HBD protein or S9.6 (anti–DNA-RNA hybrid antibody, clone S9.6; Millipore, MABE1095) was added to incubate with the bead-bound cells by rotating overnight at 4°C.

Techniques: Genome Wide

Journal: Science Advances

Article Title: Genomic profiling of native R loops with a DNA-RNA hybrid recognition sensor

doi: 10.1126/sciadv.abe3516

Figure Lengend Snippet: ( A ) Workflow of DRIPc-seq with GST-His 6 -2×HBD or S9.6 combined with random fragmentation of genomic DNA by NEB dsDNA fragmentase. IP, immunoprecipitation. ( B ) Genome browser tracks of DRIPc-seq and R loop CUT&Tag coverage at the FUS and RPL13A loci detected by GST-His 6 -2×HBD and S9.6. Signals were normalized by reads per million. ( C and D ) Heatmap analysis of DRIPc-seq (ex vivo) and R loop CUT&Tag (native, in situ) at all the protein-coding genes by GST-His 6 -2×HBD (C) or S9.6 (D). ( E and F ) Metagene plots of DRIPc-seq and R loop CUT&Tag by GST-His 6 -2×HBD (E) or S9.6 (F).

Article Snippet: Two micrograms of recombinant GST-His 6 -2×HBD protein or S9.6 (anti–DNA-RNA hybrid antibody, clone S9.6; Millipore, MABE1095) was added to incubate with the bead-bound cells by rotating overnight at 4°C.

Techniques: Immunoprecipitation, Ex Vivo, In Situ