GSM7886218: Flag_ChIP [Flag1_S2_L004]; Drosophila melanogaster; ChIP-Seq
120 late third instar larval heads, containing 240 eye discs, were dissected into ice cold PBS. Dissected heads were then fixed in 1.5% formaldehyde in PBS for 15 minutes at room temperature. Fixed heads were quenched with 0.125 M glycine + PBS solution on ice for 5 minutes. Heads were washed in ice cold wash buffer A (10 mM Hepes pH 7.6, 10 mM EDTA pH 8.0, 0.5 mM EGTA pH 8.0, 0.25% Triton X-100) for 10 minutes followed by another wash in wash buffer B (10 mM Hepes pH 7.6, 100 mM NaCl, 1 mM EDTA pH 8.0, 0.5 mM EGTA pH 8.0, 0.01% Triton X-100) for 10 minutes at 4°C. Eye discs were dissected away from the heads and placed in ice cold wash buffer B. The eye discs were pelleted by centrifugation at 800 g for 30 seconds at 4°C. Eye discs were resuspended in 1 mL of sonication buffer (50 mM Hepes pH 7.6, 140 mM NaCl, 1 mM EDTA pH 8.0, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, supplemented with proteinase inhibitors (GenDepot P3100-001)) and then transferred to a 15 ml Falcon tube. Eye discs were then sonicated on ice with a Misonix S-4000 Sonicator, 3.2 mm probe. The sonication cycle was as follows: 10 s at 65 amplitude, 30 s rest on ice, total sonication time: 3 minutes. 10 µL of 10% SDS, 100 µL 1% sodium deoxycholate, 100 µL 10% Triton X-100, and 28 µL of 5M NaCl were added to the sonicated eye discs and incubated at 4°C for 10 minutes. Sonicated eye discs were then centrifuged at maximum speed for 5 minutes at 4°C to remove any debris. Supernatant containing the sonicated chromatin was transferred to a new tube.For ChIP-seq experiments, 30 µL of antibody conjugated Protein G Dynabeads were added to the supernatant and immunoprecipitated overnight at 4°C in a tube rotator. 5 µL rabbit anti-GFP (Invitrogen A-6455) and 5 µL of mouse anti-FLAG (M2, Sigma Aldrich, F1804) antibodies were used for each ChIP-seq experiment. Beads were washed once in each of the following buffers: sonication buffer, ChIP wash buffer A (same recipe as sonication buffer but with 500 mM NaCl), ChIP wash buffer B (20 mM Tris pH 8.0, 1 mM EDTA pH8.0, 250 mM LiCl, 0.5% NP-40, 0.5% sodium deoxycholate), and finally TE buffer for 5 minutes each at 4°C. To remove crosslinks, all supernatant was replaced with 150 µL of elution buffer (50 mM Tris pH 8.0, 50 mM NaCl, 2 mM EDTA, 0.75% SDS, 20 μg/mL RNase A) and incubated overnight at 65°C. Eluted chromatin was removed from the beads and set aside and 150 µL of fresh elution buffer was added to the beads and incubated at 65°C for 30 minutes. Supernatant was pooled with previously eluted chromatin to yield ~300 µL of eluted chromatin. 60 µg of Proteinase K was added to the eluted chromatin and incubated at 37°C for 2 hours to complete the crosslink removal process. ChIP DNA was subjected to phenol-chloroform extraction and ethanol precipitation. Total input controls were treated with the same protocol as the ChIP samples, except that there were no immunoprecipitation steps. Sonicated chromatin from total input controls was directly subjected to the crosslink removal protocol followed by Proteinase K treatment, phenol chloroform extraction, and ethanol precipitation. Libraries for next generation sequencing were generated from these DNA samples. ChIP-seq libraries were prepared and paired-end sequencing (PE75) was performed with an Illumina NextSeq 500 system. Samples were sequenced to a depth of 40 million reads. All samples passed the initial quality controls on the fastQ files.