GSM7110229: S2-WT_aTwi_IP_rep2; Drosophila melanogaster; ChIP-Seq
For each ChIP experiment, ~50 million cells were crosslinked by adding methanol-free formaldehyde directly to the cell culture medium to a final concentration of 0.8%. Cells were rotated on a nutator for 7 minutes at room temperature (RT) before quenching of crosslinking by adding glycine to 125 mM. Crosslinked cells were pelleted by centrifugation at 600 × g for 3 minutes. Cells were washed twice with 1X PBS. Cells were lysed by resuspending in lysis buffer (50 mM HEPES pH 7.9, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP40, 0.25% Triton-X 100) with protease inhibitors (Pierce) and incubating on ice for 10 minutes. The resulting chromatin was pelleted for 5 minutes at 1500 × g and resuspended in RIPA buffer. Chromatin was sonicated in a Covaris S220 Ultrasonicator (5 cycles of 120 seconds with 60 second delay, 170 peak power, 10% duty factor, 200 cycles/burst). Sonicated chromatin was centrifuged for 10 minutes at 10,000 × g to pellet insoluble material. 5% of the supernatant was set aside as input, and the remainder was incubated at 4°C overnight with antibodies (6 µL anti-Zld, 8 µL anti-Grh, 10 µg anti-Twi, 7.5 µL anti-HA). 20 µL protein A beads (Dynabeads Protein A, ThermoFisher Scientific) blocked with BSA were added and samples were incubated at 4°C for 4 hours. Beads were separated on a magnet and washed 3× with low salt wash buffer (10 mM Tris pH 7.6, 1 mM EDTA, 0.1% SDS, 0.1% Na-Deoxycholate, 1% Triton-X 100, 150 mM NaCl), 2× high salt wash buffer (10 mM Tris pH 7.6, 1 mM EDTA, 0.1% SDS, 0.1% sodium deoxycholate, 1% Triton-X 100, 300 mM NaC), 2× LiCl wash buffer (0.25 M LiCl, 0.5% NP40, 0.5% sodium deoxycholate), and 1× with TE buffer with NaCl (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 50 mM NaCl). Beads were resuspended in elution buffer (50 mM Tris-HCl pH 8.0, 1% SDS, 10 mM EDTA) and incubated at 65°C for 10 minutes. The IP and input samples were treated with 4.5 µL RNAse A for 30 minutes. 5 µL Proteinase K was added and samples were incubated overnight at 65°C to reverse crosslinking. DNA was isolated by phenol:chloroform extraction, precipitated with EtOH, and resuspended in 20 µL H2O. Preparation of sequencing libraries was performed using NEBNext Ultra II kit (NEB) with 7 PCR cycles for library amplification. Sequencing was performed on the Illumina Hi-Seq4000 using 50bp single-end reads, or on the Illumina NovaSeq 6000 using 150bp paired-end reads.