Journal: Molecular cell

Article Title: Senataxin Mutation Reveals How R-loops Promote Transcription by Blocking DNA Methylation at Gene Promoters

doi: 10.1016/j.molcel.2017.12.030

Figure Lengend Snippet: Key Resources Table

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse monoclonal anti-NeuN Millipore Cat# MAB377 Rabbit monoclonal anti p-SMAD 2/3 Cell Signaling Technology Cat# 8828 Rabbit polyclonal anti-BMP2 LSBio Cat# LS-B12549 Mouse monoclonal S9.6 A gift from Dr. Stephen Leppla N/A Rabbit polyclonal anti-RNase H1 Novus Biologicals Cat# NBP2-38501 Rabbit polyclonal anti-SETX Bethyl Laboratories Cat# A301-104A-M Mouse monoclonal anti-dsDNA Abcam Cat# ab27156 Mouse IgG (For DRIP IP) Santa Cruz Cat# SC2025 Mouse monoclonal anti-Halo Promega Cat# G9211 Rabbit polyclonal anti-BAMBI ThermoFisher Cat#PA5-38027 Rabbit polyclonal anti-pSMAD2 Cell Signaling Tech.

Techniques: Control, Recombinant, Transfection, Purification, Reverse Transcription, SYBR Green Assay, Staining, Activity Assay, Methylation, DNA Methylation Assay, Mutagenesis, Sequencing, Plasmid Preparation, Software, Real-time Polymerase Chain Reaction

Journal: Nucleic Acids Research

Article Title: DDX17 helicase promotes resolution of R-loop-mediated transcription–replication conflicts in human cells

doi: 10.1093/nar/gkac1116

Figure Lengend Snippet: Loss of DDX17 induces accumulation of R-loops during S-phase. ( A ) Loss of DDX17 induces accumulation of R-loops. Top panel: schematic representation of experimental workflow. U-2 OS T-REx [RNH1(D210N)-GFP] cells grown on coverslips were transfected with indicated siRNAs for 72 h and treated with doxycycline (1 ng/ml) for last 24 h. Prior to fixation and staining with anti-PCNA antibody, cells were pre-extracted to eliminate unbound RNase H1. Bottom panel: Representative images. Nuclear foci of RNH1(D210N)-GFP indicate sites of R-loop formation. Scale bar, 10 μm. ( B ) Quantification of the number of RNH1(D210N)-GFP nuclear foci in PCNA-positive and PCNA-negative cells for indicated conditions (n ≥ 1700). Red horizontal lines indicate mean value. **** P ≤ 0.0001 (Mann–Whitney test). ( C ) Western blot analysis of extracts of U-2 OS T-REx [RNH1(D210N)-GFP] cells transfected with indicated siRNAs. ( D ) RNH1(D210N)-GFP foci accumulate in late S and G2 phase of the cell cycle in DDX17-depleted cells. Scatter plots of PCNA (y-axis) and DAPI (x-axis) intensities in individual DDX17-depleted (right) or mock-depleted (left) cells ( n ∼ 450) to determine the cell cycle phase. The color code indicates the number of RNH1(D210N)-GFP foci per nucleus. ( E ) RNA:DNA slot blot analysis of genomic DNA isolated form U-2 OS cells transfected with indicated siRNAs. Genomic DNA was treated or not with RNase H. Blots were immunostained using S9.6 and anti-ssDNA antibodies. ( F ) Quantification of S9.6 signal on slot blots represented in (E). For each sample, S9.6 signal intensity was normalized to ssDNA intensity. The resulting values are normalized to the control siRNA (siLuc) and represent mean ± SD, n = 3. ns, non-significant ( P > 0.05); * P ≤ 0.05 (One-way ANOVA). ( G ) Loss of DDX17 induces accumulation of R-loops at R-loop-prone loci in U-2 OS cells. DRIP-qPCR analyses were performed using S9.6 monoclonal antibody and genomic DNA isolated from U-2 OS cells transfected with indicated siRNAs. The R-loop-prone regions of the APOE , BTBD19 , GADD45A and RPL13A genes were examined. An R-loop-free SNRPN gene locus was used as a negative control. Where indicated, the genomic DNA was treated with RNase H to remove RNA:DNA hybrids. Data are plotted as a percentage of the input, and represent the mean ± SD, n = 3. ( H,I ) DDX17 associates with R-loops. ( H ) U-2 OS cells were pre-extracted and sonicated to generate chromatin fragments with DNA size ranging from 800–200 bp. After centrifugation, sonicated cell lysates were immunoprecipitated with control IgG or S9.6 antibody followed by western blot analysis. ( I ) as in (H) except that, before immunoprecipitation, cell lysates were supplemented with 3 mM MgCl 2 and incubated with RNaseH for 45 min at 37°C as indicated.

Article Snippet: DRIP-derived samples were subjected to quantitative real-time PCR (qPCR) analysis using iQ SYBR Green Supermix reagent (Bio-Rad) and the gene-specific primers described below on a CFX96 Real-Time PCR detection system (Bio-Rad).

Techniques: Transfection, Staining, MANN-WHITNEY, Western Blot, Dot Blot, Isolation, Control, Negative Control, Sonication, Centrifugation, Immunoprecipitation, Incubation