title: vgn28_68h_rep2_K27ac
organism: Drosophila melanogaster
description: Protocols: The virginizer flies of 5-10 days age were transferred into fresh bottles and left to lay embryos for about 24 hours at 20C before the adults were removed from the bottles. The bottles were heat-shocked inside a 38C water bath for 1 hour. The virgins eclosed from heat-shocked bottles of the virginizer line were collected, mated to the hand-picked males from the paternal lines by putting all flies into cylindrical fly houses in a 25C room. The flies were left in the cages for 2 days before starting the collections. On the day of collection, one hour prelays were done for 3 times, followed by 2-hour collections. The plates with embryos were aged to the stages desired at 25C. The embryos were washed onto the set of sieves and dechorionated by incubating in the 50% bleach for 2.5 min. Around 200 embryos were transferred into an eppendorf tube and snap-frozen in liquid nitrogen for RNA assays. The majority of embryos were fixed by incubating in 9.5 ml cross-linking solution with 485 microliters 37% formadehyde with vigorous shaking for 15 min. Cross-linking was stopped by incubating with Glycine solution with vigorous shaking for at least 1 min. After washing with PBT, the embryos were blotted dry. The cross-linked dry embryos were transferred into 1.5 mL eppendorf tubes, snap-frozen in liquid nitrogen and kept in -80C for ATAC-seq and iChiP. 40.000 frozen nuclei in a total volume of 10 ul PBS + 0.5% SDS were used per sample. The nuclei were sonicated in a 0.1 ml sonication tube at 4C for 40 cycles 30s/30s ON/OFF using the Bioruptor Pico. For the first IP, Chromatin was immobilized with 1.3 ug of anti-H3 antibody and Dynabeads Protein G on a rotating wheel overnight at 4C. Following Chromatin End Repair and A-tailing, the samples were indexed by ligation of Y-shaped Index Adaptors containing P5 and P7 sequences. After Chromatin Release, samples were pooled by 8, resulting in three pools. These pools were split into two second IPs each. The chromatin was incubated at 4C for 3h on a rotating wheel with 2.5 ug of antibody against H3K27ac or H3K4me3, followed by a 1 h incubation with Dynabeads Protein G. The IPs were washed, the ChIPed DNA was eluted and purified. KAPA HiFI Hot Start Ready Mix and primers containing the Illumina P5-Read1 and P7-Read2 sequences were used following instructions. After a final clean-up with AmPure XP beads (Beckman, Cat No. 63880), the quality of the libraries was determined by qPCR. To assess efficiency and specificity of the ChIPs, we used a positive and a negative target region and compared the enrichments before and after library amplification. The libraries were run on a Bioanalyzer chip, multiplexed and sequenced with Illumina platform.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2020-12-04T04:07:44Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2020-03-05T13:58:11Z
attributes.tag: External Id
attributes.value: SAMEA6605276
attributes.tag: INSDC center name
attributes.value: EMBL
attributes.tag: INSDC first public
attributes.value: 2020-12-04T04:07:44Z
attributes.tag: INSDC last update
attributes.value: 2020-03-05T13:58:11Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-8880:vgn28_68h_rep2_K27ac
attributes.tag: age
attributes.value: 6-8h
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: common name
attributes.value: fruit fly
attributes.tag: developmental stage
attributes.value: stage 11
attributes.tag: initial time point
attributes.value: egg laying
attributes.tag: sample name
attributes.value: E-MTAB-8880:vgn28_68h_rep2_K27ac
attributes.tag: scientific_name
attributes.value: Drosophila melanogaster
attributes.tag: sex
attributes.value: mixed sex
attributes.tag: strain
attributes.value: vgn x DGRP 28