title: GSM8072473: GFPKD, Pol2s5p, rep2; Drosophila melanogaster; ChIP-Seq
library_construction_protocol: 50-100 pairs of ovaries were dissected in 1x PBS, then fixed in 1% Formaldehyde solution for 10 min at room temperature with rotation. Then, ovaries were quenched with 1/16th volume of 2.5M Glycine for 5 min with rotation, washed in 0.2% TritonX-100 in PBS x3 for 5 min each, supernatants were removed, and samples were stored at -80°C freezer or proceeded for next steps. The fixed ovaries were gently homogenized in 1x PBS with polypropylene pestle and spun down at 800 g for 1 min. Supernatants were removed and ovaries were resuspended in 200 µl ChIP lysis buffer [10 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Na-Deoxycholate, 0.5% N-laurolysarcosine, 1mM  DTT, 1x protease inhibitor] and transferred to 1.5 ml TPX Plastic Micro Tube (Diagenode) for 10 min sonication using Bioruptor at 30 s ON and 45 s OFF intervals. Sonication was completed when majority of DNA smears were at 200-500 bp sizes. Sonicated chromatins were incubated with 30 µl of Dynabeads Protein G (Dynabeads) for 1 hr at 4°C with rotation to pre-clean the sample. Then, tubes were placed on the magnet and supernatants were transferred to new 1.5 ml tubes. Appropriate antibodies were added into tubes with pre-cleaned chromatin (4 µg of rabbit IgG, or rabbit anti-Pol II Ser5P (ab5131), or rabbit anti-Pol II Ser2P (ab5095)) and incubated overnight at 4°C with rotation. 5% of chromatins were saved as input samples and remaining samples were incubated with 60 µl of Dynabeads for three hr at 4°C with rotation. Beads were washed four times with 0.2% TritonX-100 in PBS for five min each and washed once with TE buffer (10 mM Tris, 0.1 mM EDTA). Tubes were placed on the magnet, supernatants were removed, and chromatin were eluted with 200 µl of Elution Buffer [50 mM Tris-HCl, pH8.0, 10 mM EDTA, 1% SDS] by incubating at 65°C for 15 min. Beads were captured by the magnet and the supernatant from each sample was collected as the immunoprecipitated chromatin. Reverse crosslinking of chromatins was performed by incubating samples at 65°C overnight with 20 µg RNaseA (ThermoFisher) and 40 µg Proteinase K (ThermoFisher). The DNA was purified using the ChIP DNA Clean & Concentrator kit (Zymo). ChIP-seq library was prepared by the Yale Stem Cell Center Genomics and Bioinformatics Core