title: GSM6737976: H3K4me3, brat_null-fruC::myc, CUT&RUN 2; Drosophila melanogaster; OTHER
library_construction_protocol: Larval brains were dissected in 45-min time windows in PBS and transferred to 0.5mL Eppendorf tubes. Dissected brains were then collected at the bottom of the tube, and supernatant was removed. CUTANATM  ChIC/CUT&RUN (Epicypher) was performed per the manufacturer's protocol, with modifications. Brains were resuspended in 100µL wash buffer, and then homogenized by ~30-50 passes of a Dounce homogenizer. Homogenized samples were transferred to a 1.5mL Eppendorf tube, and then the cells were pelleted by centrifugation (600g for 3min). Next, then pellets were processed using the CUT&RUN kit.  A total of 0.5ng of antibody was used per sample, (or 0.5µL if antibody concentration was unknown). A total of 0.5 ng of Escherichia coli spike-in DNA was added into each sample as a spike-in control.     For control brains, antibodies used were goat anti-cMyc (abcam, ab9132), mouse anti-Su(H), rabbit anti-Trl, rabbit anti-Caf1 (Gift from J. Kadonaga, (Tyler et al., 1996)), rabbit anti-Su(z)12 (Gift from G. Cavalli, (Loubière et al., 2016)), rabbit anti-IgG (Epicypher), and rabbit anti-H3K9me3 (abcam, ab8898). For both control and fru-/- brains, antibodies used were rabbit anti-H3K4me3 (Active Motif: 39159), rabbit anti-H3K27me3 (Sigma Aldrich, 07-449), and rabbit anti-H3K27ac (Active Motif: 39136). A total of 50 brains were collected for transcription factor samples and 25 brains were collected for histone mark samples. All samples were performed in duplicate. Samples targeting acetylation had 100 mM of sodium butyrate (Sigma Aldrich) added to all buffers. Samples using mouse antibodies underwent an additional antibody incubation step, where samples were washed 2x with cell permeabilization buffer after primary antibody incubation, and then were incubated for 1 hr with 0.5ng of rabbit anti-mouse IgG. Fragmented DNA was diluted to 50µL in 0.1X TE and library prepped using the NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® (E7645) with NEBNext® Multiplex Oligos for Illumina (E6440) per the manufacturer's protocol with modifications. The adaptor was diluted 1:25, and bead clean-up steps were performed using 1.1x AMPure Beads without size selection. The PCR cycle was modified to match specifications provided by the CUTANATM  ChIC/CUT&RUN kit. DNA was eluted in 20µL 0.1X TE. DNA samples were assessed for concentration and quality on an Agilent TapeStation. Samples with greater than 1% adaptor underwent an additional round of bead cleanup. Samples that passed quality control were sequenced on an IlluminaNovaSeq-6000 chip (University of Michigan Advanced Genomics Core). Then, 151-bp paired end sequencing was performed, with a target of at least 10,000,000 reads per replicate.