title: GSM8746388: Drosophila,wingdisc,H2Av_EGFP2_1(ChIP); Drosophila melanogaster; ChIP-Seq
library_construction_protocol: The ChIP experiments were performed following previous protocols with minor modifications. Wing imaginal discs were collected and transferred into a 1.5 ml loBind Eppendorf tube (022431021, Eppendorf).  Ice-cold buffer A1 was added and tissues were lysed for 5 min.  The samples were subjected for cross-linking in 1% formaldehyde for 15 min at RT, which were ended by adding of 160 ul 2.5 M glycine and rotating for 5 min. Chromatin fragmentation was performed with the Bioruptor sonicator (JY92II, Xinchen Biotech, Nanjing, China) using a high-power program (400W power, ON = 10 sec, OFF = 30 sec, 10 times). After sonication, the lysate was centrifuged and the supernatant was transferred into a new 1.5ml LoBind tube. The amount of DNA in each sample was quantified using the Equalbit 1× dsDNA HS Assay Kit (EQ121-00, Vazyme). The DNA samples were pre-cleared with 15 µl IgG and 15 µl protein A+G Dynabeads (10001D, Invitrogen) for 2 h at 4℃. Each sample was then incubated with 5 µg antibody for 4 h at 4℃, after which 30 µl Dynabeads were added and overnight incubation was done with rotating. After five washes in buffer A2 and a final wash in TE, the samples were eluted with 250µl elution buffer and de-crosslinked at 65°C for 6 h. Protease K was used to degrade proteins in the elution, after which the DNA was precipitated in -20 ℃ by adding 1/10 volume of 3M NaAc (pH5.5), 2 µl of 35 mg/ml glycogen and 2.5 volumes of 100% EtOH. DNA pellet was air dried and dissolved in the required volume of nuclease free H2O. For qPCR, we used 5 µl of Perfect Start Green qPCR Super Mix (AQ602, TransGen Biotech, Beijing, China), 0.1 µl of each primer (50 pmol/L), 2.8 µl of ddH2O and 2 µl of DNA sample. The real-time quantitative PCR experiments were conducted on ABI QuantStudio 6 Flex System (Thermo Fisher). Primer sequences for E(spl)-C genes were obtained from (Pillidge and Bray, 2019), and sequences of primers used in this study are listed in Supplementary Table 1. The value of % Input was calculated by the following formula: % Input = 100* 2^ (-(Ct [ChIP] - (Ct [Input] -Log2 (Input Dilution Factor)))). Antibodies used for ChIP include rabbit anti-H2Avac (ab232908, Abcam), mouse anti-H2Av (61751, Active Motif) and rabbit IgG (2729, Cell Signaling) for mock control. Buffer A1 contains 60 mM KCl, 15 mM NaCl, 4 mM MgCl2, 15 mM HEPES, 0.5% Triton X-100, 0.5 mM DTT and 10 mM sodium butyrate. Buffer A2 contains 140 mM NaCl, 15 mM HEPES, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.5 mM DTT, 0.05% SDS, 0.1% sodium deoxycholate and 10 mM sodium butyrate. Elution buffer contains 10mM EDTA, 50mM Tris HCl, 1% SDS. The purified DNA was used for ChIP-seq library preparation. The library was constructed by Novogene Corporation (Beijing, China). Subsequently, pair-end sequencing of sample was performed on Illumina platform (Illumina, CA, USA). Library quality was assessed on the Agilent Bioanalyzer 2100 system