Journal: Epigenetics & Chromatin
Article Title: 27nt-RNAs guide histone variant deposition via ‘RNA-induced DNA replication interference’ and thus transmit parental genome partitioning in Stylonychia
Figure Lengend Snippet: Results of ‘RNA-induced DNA replication interference’ assays. a Effects of 27nt-RNAs using Pfu or Taq polymerases after end-point-PCR and agarose gel electrophoresis (top) or after qPCR (bottom). b Effects of 27nt-RNAs alone or in combination with PIWI1 on linear DNA amplification in a Klenow reaction were assayed via qPCR. c Hypothetical models on sequence-specific targeting through Argonaute/PIWI-RNA complexes (blue: IES, red: MDS, yellow: PIWI1, green: 27nt-RNA, orange: tethered transcript [ c1 only]): c1 According to the ‚nascent transcript model, 27nt-RNA/PIWI1 complexes could target tethered IES-originating transcripts, which would be reminiscent of observations made in divergent eukaryotes, such as S. pombe , C. elegans and A. thaliana ]). Alternatively, we assumed that 27nt-RNA/PIWI1 complexes could interact with dsDNA ( c2 ) or via base-pairing with ssDNA, possibly when it occurs in a replication bubble ( c3 ). d Mapping of mRNA reads purified 20 h PC on micronuclear model genes reveals that IES (red bars) are sharply omitted
Article Snippet: Standard PCR conditions were as follows: 95 °C for 10 min, 30 cycles of [95 °C for 15 s, 60 °C for 30 s, 72 °C for 30 s], 72 °C for 10 min; alternatively, for the 856 bp PCNA amplicon the conditions were: 95 °C for 15 min, 30 cycles of [95 °C for 15 s, 60 °C for 30 s, 72 °C for 45 s], 72 °C for 10 min. Quantitative PCR was performed on a Rotor Gene 6000 (Corbett Life Science) using QuantiTect SYBR green Taq polymerase Master Mix (Qiagen) or Pfu DNA polymerase (ThermoFisher) and SYBR green (Sigma-Aldrich).
Techniques: Polymerase Chain Reaction, Agarose Gel Electrophoresis, Real-time Polymerase Chain Reaction, Amplification, Sequencing, Purification