GSM4745381: ChIP-Pol-S2P_DMSO_mock-RNAi_S2; Drosophila melanogaster; ChIP-Seq
Crosslinking was performed with 1% formaldehyde for 10 min at RT and stopped by adding glycine (crosslinking in Drosophila tissues was performed by 0,7% formaldehyde). After washings, chromatin was lysed in SDS-containing buffer (50 mM HEPESKOH, pH 7.9; 140 mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% Na deoxycholate, 0.1% SDS) with protease inhibitors cocktail (Roche) and sheared to 500-2000 bp fragments by sonication. Sonicated chromatin was centrifuged twice at 16 000 g for 20 min, and used in immunoprecipitation experiments. For one experiment, about 5 ug of antibodies and 8 uL of MabSelect sepharose (GE healthcare) were taken; BSA were added to a final concentration of 1%. The precipitated chromatin was sequentially washed twice with SDS-containing buffer and with TE buffer (20mM Tris-HCl, pH 8.0; 1mM EDTA). Precipitated chromatin complexes were eluted by sequential incubation in two portions of elution buffer (50mM Tris-HCl, pH 8.0; 1mM EDTA, 1% SDS), 30 min each, at room temperature. The eluted chromatin solution was supplemented with 5 M NaCl (16 uL per 500 uL sample) and incubated at 65°C for 16 h on a thermoshaker for de-crosslinking. De-crosslinked chromatin was subjected to proteinase treatment (3 uL of proteinase K and 5 uL of 0.5M EDTA per 500 uL sample) and incubated at 55°C for 4 h on thermoshaker. DNA was extracted with a phenol/chloroform mixture and precipitated with isopropanol. The precipitate was dissolved in TE buffer and subjected to library preparation. DNA libraries were prepared for sequencing using standard Illumina protocols