title: vgn639_24h_rep1_ATAC
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. Frozen embryos were transferred into a 7 mL douncer placed on ice, thawed in 4 mL HB buffer with proteinase inhibitor. Embryos were dounced 20 times with the loose pestle and 10 times with the tight pestle. The homogenate was filtered through two layers of Miracloth and centrifuged for 7 min at 3500 rpm at 4C and the supernatant was discarded. The nuclei pellet was resuspended in 3 mL HB buffer and transferred to a clean tube. The sample was centrifuged for 7 min at 3500 rpm at 4C and the supernatant was discarded. The nuclei pellet was resuspended in 3 mL PBT and transferred to a clean tube. The nuclei were dissociated by passing them 10 times through a 20G needle and 10 times through a 22G needle using a 5 mL syringe and then filtered through 20 ??m Nitex membrane on a funnel into a clean 15 mL tube. The nuclei extracted were quantified using the LSRFortessa system. 200,000 nuclei in 50 microl PBT was supplemented with 50 microl permeabilization solution and incubated on a nutator for 30 min at 4C. The nuclei were centrifuged at 3200 g for 5 min at 4C and supernatant removed. The nuclei pellet was resuspended in 50 microl tagmentation mix, containing 5 microl TDE1, 25 microl 2x tagmentation buffer (both from Nextera kit) and 20 microl nuclease-free water, and incubated at 37C for 30 min. 50 microl STOP buffer and 5 microl of 1 mg/mL RNase A were added to stop the reaction and digest RNA by incubating on a thermomixer at 55C for 10 min with 900 rpm shaking. The reverse cross-linking and protein digestion were done by adding 3 microl of 20 mg/mL Proteinase K and incubating at 65C overnight with 900 rpm shaking. The reaction was purified using the MinElute column accompanied with Qiagen PCR purification buffers. The DNA concentration was measured with Qubit. 20 ng of tagmented DNA was brought to 10 microl with water. 2.5 microl i7 primer, 2.5 microl i5 primer, 2.5 microl Nextera primer cocktail and 7.5 microl Nextera PCR mastermix (all from Nextera kits) were added to complete the PCR reaction. PCR was run using the following program: 72C, 3 min; 98C, 30 sec; cycle start: 98C, 10 sec, 63C, 30 sec, 72C, 3 min, cycle close (run 12 cycles); Hold at 10C. The PCR product was run on a 1.2% agarose gel with 100 bp DNA ladder. Gel piece from 100 bp to 1 kb was cut with SafeXtractor on a blue light transilluminator. DNA was extracted from the gel using Qiagen gel extraction kit and MinElute columns.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2020-12-04T04:07:45Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2020-03-05T13:18:22Z
attributes.tag: External Id
attributes.value: SAMEA6605139
attributes.tag: INSDC center name
attributes.value: EMBL
attributes.tag: INSDC first public
attributes.value: 2020-12-04T04:07:45Z
attributes.tag: INSDC last update
attributes.value: 2020-03-05T13:18:22Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-8879:vgn639_24h_rep1_ATAC
attributes.tag: age
attributes.value: 2-4h
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: common name
attributes.value: fruit fly
attributes.tag: developmental stage
attributes.value: stage 5
attributes.tag: initial time point
attributes.value: egg laying
attributes.tag: sample name
attributes.value: E-MTAB-8879:vgn639_24h_rep1_ATAC
attributes.tag: scientific_name
attributes.value: Drosophila melanogaster
attributes.tag: sex
attributes.value: mixed sex
attributes.tag: strain
attributes.value: vgn x DGRP 639