Genetic Manipulation and RNA-DNA Detection

Endogenous genes were deleted as described (23 (link)). Yeast strains and primers used are listed (Supplementary Tables S3 and S4). R-loop detection via direct pull downs employed the anti-RNA–DNA antibody from Kerafast (cat# ENH001), which exhibited lower enrichments but higher specificity compared to antibodies that we used previously in chromatin immunoprecipitation (ChIP) (12 (link)). Anti-RNAPII, anti-diAcH3K9/14, anti-TAP and anti-β-Actin antibodies were purchased from BioLegend (665004), Millipore (06-599), Sigma (P1291-500UL) and Thermo Scientific (MA1-744), respectively. Anti-GFP was a kind gift from D. Moazed (Harvard University). The Stm1 YEplac181 plasmid or empty vector control were kind gifts from F.B. Johnson (University of Pennsylvania) (24 (link)).

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Abraham K.J., Chan J.N., Salvi J.S., Ho B., Hall A., Vidya E., Guo R., Killackey S.A., Liu N., Lee J.E., Brown G.W, & Mekhail K. (2016). Intersection of calorie restriction and magnesium in the suppression of genome-destabilizing RNA–DNA hybrids. Nucleic Acids Research, 44(18), 8870-8884.

Publication 2016

anti-β-Actin MA1-744

Actin Anti antibodies Anti dna antibody Antibodies Chromatin immunoprecipitation Genes Gifts Loop Plasmid Primers R loop Rnapii Strains Yeast

Corresponding Organization :

Other organizations : Canada Research Chairs, University of Toronto

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Variable analysis

independent variables

Endogenous genes deletion
Stm1 YEplac181 plasmid or empty vector control

dependent variables

R-loop detection via direct pull downs

control variables

Yeast strains
Primers used
Anti-RNA–DNA antibody from Kerafast
Anti-RNAPII, anti-diAcH3K9/14, anti-TAP and anti-β-Actin antibodies from BioLegend, Millipore, Sigma and Thermo Scientific, respectively
Anti-GFP antibody from D. Moazed (Harvard University)

controls

Positive control: Not specified
Negative control: Empty vector control

Annotations

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