GSM1462847: strain9-Input-Rep2; Drosophila virilis; ChIP-Seq
Flies were put on yeast for 2-3 days and 100 ovaries per IP condition were dissected. Ovaries were fixed for 10 min at RT using 1% paraformaldehyde (PFA) followed by 5 min quenching at RT by directly adding glycine (final concentration 25mM) and 3 washes in PBS. Ovaries were slightly dounced in Farnham Buffer (5mM HEPES pH8.0; 85mM KCl; 0.5% NP-40/Igepal; NaCl Protease Inhibitor Cocktail; NaF 10mM; Na3VO4 0.2mM), palleted, followed by strong douncing in RIPA Buffer (20mM Tris pH7.4; 150mM; 1% NP-40/Igepal; 0.5% Sodium Deoxycholate; 0.1% SDS; Protease Inhibitor Cocktail; NaF 10mM; Na3VO4 0.2mM) prior to sonication using a Bioruptor from Diagenode on medium power for 20 cycles (30sec ON, 30sec OFF). Samples were then centrifuged, supernatant collected and pre-cleared for 2h at 4ºC using Dynabeads Protein G (Invitrogen). Antibodies were conjugated to Dynabeads Protein G for 2h at 4ºC. A sample of 5% of the pre-cleared supernatant was saved as input control fraction and the rest was incubated with the antibody-conjugated beads for 2h at 4ºC. The beads were washed 5 times at 4ºC using LiCl IP Buffer (10mM Tris ph7.5; 500mM LiCL; 1% NP-40/Igepal; 1% Sodium Deoxycholate), once with TE. Samples were digested in Proteinase K Buffer (200mM Tris ph7.4; 25mM EDTA; 300mM NaCL; 2% SDS) with 100µg of proteinase K. Samples for 3h at 55ºC and revers-crosslinked overnight at 65ºC. DNA was extracted following standard phenol-chloroform extraction and concentration was measured by Qubit. Libraries were generated following Illumina's DNA Sample Prep Kit protocol