title: GSM4889964: M1_d1_15_M2_MSL3; Drosophila melanogaster; ChIP-Seq
library_construction_protocol: For immunoprecipitation chromatin was pre-cleared with Protein A Dynabeads (Thermo Fisher), blocked with 0.5% BSA. Samples containing 20 µg of DNA equivalent in 0,5 mL of nuclear lysis buffer were incubated overnight, at 4°C on a rotary shaker, with rabbit antibodies against MSL1 (1:300), MSL2 (1:200), MSL3 (1:200), or with nonspecific IgG purified from rabbit preimmune sera (control). Chromatin–antibody complexes were collected using blocked Protein A Dynabeads (Thermo Fisher) at 4°C over 5 h. After three rounds of washing with nuclei lysis buffer supplemented with 500 mM NaCl, and TE buffer (10 mM Tris-HCl, pH 8; 1 mM EDTA), the DNA was eluted with elution buffer (50 mM Tris-HCl, pH 8.0; 1 mM EDTA, 1% SDS), treated with RNase A for 30 min and proteinase K overnight, incubated at 65 °C for 6 hours and extracted  with ChIP DNA Clean&Concentrator (Zymo Research). The ChIP-seq libraries were prepared with NEBNext® Ultra™ II DNA Library Prep kit, as described in the manufacturer's instructions. In short, eluted DNA was end-repaired and terminal adenosine residues were added using the NEBNext reagents. Indexed adapters were ligated, after which the material was size selected at ~200-600 bp with Ampure XP beads (Beckman Coulter). PCR amplification was performed using NEB primers for 15-16 cycles using the Q5 Hot Start HiFi PCR Master Mix (NEB). The PCR-amplified library was purified using Ampure XP beads and its quality was assessed on a Bioanalyzer 2100 system (Agilent). Diluted libraries were clustered on a pair-read flowcell and sequenced using a NovaSeq 6000 system (Illumina, United States).