ChIP-seq across 4 Transcription Factors and 3 time-points in 10 Drosophila crosses of inbred lines
ChIP-seq across 4 Transcription Factors and 3 time-points in 10 Drosophila crosses of inbred lines
For F1 crosses, 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. For samples from parental lines, males and females were counted to match the numbers of the F1 crosses. The flies were put into cylindrical fly houses in a 25C room.Â All 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. Embryos were fixed as described in Sandmann et al. 2006 (doi: 10.1038/nprot.2006.383) Frozen embryos were transferred into a 7 mL douncer placed on ice, thawed in 4 mL HB buffer with proteinase inhibitor; 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 micrometer Nitex membrane on a funnel into a clean 15 mL tube. The frozen nuclei pellet was resuspended with an appropriate amount of RIPA buffer, and incubated on ice for 10 min. The nuclei were then sonicated in the Bioruptor Pico according to the manufacturer's instructions for 15 cycles (30 sec on / 30 sec off). The supernatant was transferred into 1.5ml low binding tubes (Eppendorf, 0030108051) and centrifuged at 20.000 xg for 10 min at 4C. Aliquots of chromatin were then snap-frozen in liquid N2 and stored at -80C. Chomatin IP was performed as described in Bonn et al. 2012 (doi: 10.1038/ng.1064) ChIP libraries were prepared using the NEXTflex qRNA-Seq Kit v2 for Illumina platforms following the Option 2 in the manufacturer's protocol.