BioProject

A total of 16 pupal wings were dissected from two females and two males at 40% pupal development for 2 library replicates, and similarly 16 pupal wings from 4 individuals at 60% pupal development for 2 library replicates. Tissues from each individual were fixed in 1% formaldehyde in PBS, before adding PBS with 0.135 M glycine for 5 min, then washing with ice-cold PBS twice. All liquid was removed prior to flash-freezing tissues in liquid nitrogen for 30 s. We performed sonication and library preparation for ChIP-sequencing following previous recommendations. Fixed tissues were dissociated in a sucrose buffer supplemented with Protease Inhibitor (PI) using a dounce homogenizer. Homogenized tissues were then spun at 2,000 g for 5 min, and the cell pellet was treated with 1 mL freshly prepared ATAC lysis buffer and PI for 5 min. During this treatment, the cell suspension was pipetted to avoid clumping. Cells in the lysis buffer were spun at 2,000 g for five minutes. The lysis buffer was removed, and the pellet was resuspended with 1,000 mL of 1x ChIP Dilution Buffer (Cell Signaling). 150-200 uL of the cell suspension was then distributed into Diagenode 1.5 mL TPX microTubes for sonication. Cell suspensions were then sonicated in < 4C in a Diagenode Bioruptor UCD-200 (3 x 5 min, 30 s on with high setting, 30 s off). Sonicated cell suspensions were then centrifuged at 12,000 g to release chromatin, then supernatants from individual tubes were pooled for 20-50 ug of DNA per 1 mL. Sheared DNA was then treated overnight with 7 uL of 0.550 mg/mL anti-Bab antibody at 4C. 1-5 ug sheared DNA was separated for negative control and incubated at 4C overnight. Both control and treatment sheared DNA were then treated with 10 uL of Dynabeads Protein A and 10 uL of Protein G (ThermoFisher Scientific Inc. 10001D, 10003D) for a minimum of 2 hours nutating at 4C. A magnetic rack was used to separate the immunoprecipitated DNA on Dynabeads, washing three times with 1,000 uL of 1x ChIP dilution buffer and twice with 1,000 uL of 1x ChIP Dilution Buffer supplemented with 70 uL of 5M NaCl. Immunoprecipitated DNA on magnetic beads were then resuspended in 1x 150 uL of ChIP Elution Buffer (Cell Signaling Technology) and incubated for 1.5 h at 65C vortexing every 10 min. After 1.5 h, the supernatant containing the immunoprecipitated DNA was treated with 2 uL of 5M NaCl and 10 uL ProteinaseK, then incubated for 2.5 h to de-crosslink. The samples were then purified using the SimpleChIP Chromatin IP kit (Cell Signaling Technology). DNA libraries were made by normalizing the input to the ChIP-ed DNA, using a NEBNext Ultra Prep II DNA library kit without size selection and with 13 cycles of PCR amplification. Prepared libraries were cleaned with 0.9x Ampure beads and checked for fragment distribution, before sequencing by the Biotechnology Resource Center (BRC) Genomics Facility (RRID:SCR_021727) at the Cornell Institute of Biotechnology on their Illumina NextSeq 500/550 platform. ChIP-seq libraries were sequenced as PE42 and PE37 reads for samples at 40% and 60% pupal development, respectively.