Massive sequencing of hybrid DNA obtained by DNA:RNA immunoprecipitation (DRIP) in extracellular microvesicles (MV) produced by HeLa cells (using S9.6 antibody)

Hybrid (Bound) and non-hybrid (Unbound) DNA from Hela EV was isolated using DRIP procedure as follows. Between 1-5µg of nucleic acid DNA from EVs (DNase0 treated) was incubated overnight with 1µg of anti-DNA-RNA Hybrid (S9.6) antibody (KeraFast) under rotation at 4°C in 500µl of binding buffer (10mM NaPO4 pH 7.0, 140 mM NaCl, 0.05% Triton X-100). The following day, 25µl of A/G magnetic beads (Pierce™) were washed twice in 500µl of binding buffer (30' min each wash) and added for 2h at room temperature under rotation to the S9.6+nucleic acid complex. The nucleic acids bound to the S9.6 antibody (Hybrid fraction or Bound) were separated from those unbound (Non-Hybrid or Unbound) by using a magnetic rack (which captured the Bead+Hybrid fraction). 500µl of the unbound fraction (bead-free) were transferred into a new 1.5ml tube and collected for further analysis. The Bead+Hybrid fraction was washed twice in 250µl binding buffer for 15' min at room temperature under rotation. The hybrids were then eluted with 250µl of elution buffer (50mM TRIS pH 8.0, 10mM EDTA, 0.5% SDS) for 15' min at room temperature under rotation. The elution step was repeated twice to make sure that all the hybrid molecules were collected. As a control of all there experiments to make sure, we were isolating and precipitating Hybrid DNA:RNA molecules, samples were also pre-treated with RNaseH1 (RNaseH), which degrades the RNA filament only when present in the hybrid structure.