GSM1440371: Cutoff PFA ChIPseq; Drosophila melanogaster; ChIP-Seq
100 flies, per IP condition, were put on yeast for 2 to 3 days before ovary dissections. Ovaries were fixed using Paraformaldehyde (PFA) at a final concentration of 1% and incubated for 10min at RT. Samples were quenched by adding directly glycine (finale concentration 25mM) for 5min at RT, and then washed 3 times in PBS. Ovaries were afterwards 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) 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. Sonication was done using a Bioruptor from Diagenode on Medium power for 20 cycles (30sec ON, 30sec OFF). Samples were centrifuged, supernatant collected and pre-cleared for 2h at 4C using Dynabeads Protein G (Invitrogen) beads. If RNase treatment was required, half of the sample was incubated with 1µl of RNase A (10mg/ml) during pre-clearing and all samples incubated 2h at RT. Meanwhile, in parallel, antibodies were conjugated to Dynabeads Protein G for 2h at 4C too. 5% of pre-cleared samples were saved for Input fraction and the rest was then transferred to the antibody-conjugated beads and incubated for 2h at 4C. Beads were then washed 5 times at 4C using LiCl IP Buffer (10mM Tris ph7.5; 500mM LiCl; 1% NP-40/Igepal; 1% Sodium Deoxycholate), then rinse in TE and finally resuspended in Proteinase K Buffer (200mM Tris ph7.4; 25mM EDTA; 300mM NaCL; 2% SDS) with 100µg of proteinase K. Samples were incubated 3h at 55C then overnight at 65C. DNA was then extracted following standard phenol-chlorophorm extraction and concentration was measured by Qbit. In case of the Cutoff ChIP-seq, the experiment was also performed using an altered protocol: ovaries were treated with Collagenase (2mg/ml) for 3 minutes at room temperature (RT), wash once in PBS, then fixed using fresh EGS (Thermo Scientific, 21565) solution (1,5mg/ml final in PBS) for 30 min at RT. Paraformaldehyde (PFA) was then added to the solution to a final concentration of 1% and incubated for 10min at RT. Consecutive steps were as previously described. NEBNext ChIPseq Library Kit
GEO Accession
GSM1440371