title: GSM5510284: Adult Brain, CUT TAG Dros Ey IgG Ab; Drosophila melanogaster; OTHER
library_construction_protocol: D. melanogaster adult brains were dissected and transferred to a tube containing 100 µl ice cold DPBS solution. After centrifugation at 800 g for 5 min, the supernatant was replaced by 500 µl nuclei lysis buffer composed of 10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20, 0.1% Nonidet P40, 0.01% Digitonin, and 1% BSA, in Nuclease-free water. The following procedure was followed to extract the nuclei from the brain tissue: incubation in nuclei lysis buffer on ice for 5 min, transfer to a dounce tissue grinder tube (Merck), 25 strokes with pestle A, incubation on ice for 10 min, 25 strokes with pestle B. The lysis was stopped by adding 1 ml of wash buffer composed of 10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20 and 1% BSA, in Nuclease-free water. Nuclei were pelleted by centrifugation at 800 g for 5 min at 4°C and resuspended in 1x Nuclei Buffer (10x Genomics). Nuclei suspensions were passed through a 40 µM Flowmi filter (VWR Bel-Art SP Scienceware). Nuclei concentration was assessed by the LUNA-FL Dual Fluorescence Cell Counter. After centrifugation, supernatant was removed and isolated nuclei were resuspended in nuclear extraction (NE) buffer (EpiCypher CUT&Tag Protocol v1.5). Nuclei concentration was assessed by the LUNA-FL Dual Fluorescence Cell Counter. 100,000 nuclei in 100 µl NE buffer were used for each CUT&Tag reaction. All subsequent steps were followed according to the EpiCypher CUT&Tag Protocol v1.5. For each reaction, 11 µl of BioMag Plus Concanavalin A beads (ConA beads, Gentaur, Cat 86057-3) were washed twice with 100 µl cold Bead Activation buffer and then resuspended in 11 µl of cold Bead Activation buffer. To bind nuclei to the activated ConA beads, 100 µl of nuclei and 10 µl of activated ConA beads were incubated for 10 min at room temperature. After supernatant removal, nuclei-bead slurry was resuspended in 50 µl Antibody150 buffer. 0.5 µg of primary rabbit anti-GFP antibody (Abcam, Cat ab290) was added per sample and incubated overnight at 4°C. Beads were cleared and resuspended in 50 µl cold Digitonin150 buffer. 0.5 µg of anti-rabbit secondary antibody (EpiCypher, Cat 13-0047) was added per sample and incubated for 30 min at room temperature. While on a magnet, beads were washed twice with 200 µl cold Digitonin150 buffer. Beads were resuspended in cold Digitonin300 buffer and incubated for 1 hour at room temperature with 2.5 µl CUTANA pAG-Tn5 (EpiCypher, Cat 15-1017). To initiate tagmentation reaction, beads were resuspended in 50 µl cold Tagmentation buffer and incubated for 1 hour at 37°C. Beads were resuspended in 50 µl TAPS buffer and 5 µl SDS Release buffer was added to quench the tagmentation reaction. To release tagmented chromatin fragments into solution, samples were incubated for 1 hour at 58°C. 15 µl SDS quench buffer was added per sample. To amplify tagmented chromatin fragments, 2 µl each of individual barcoded P5 and P7 sequencing adapters (10 µM stock) and 25 µl non-hot start CUTANA High Fidelity 2x PCR Master Mix (EpiCypher, Cat 15-1018) were added and 18 cycles of CUT&Tag-specific PCR parameters were used (58°C for 5 min, 72°C for 5 min, 98°C for 45 sec, 98°C for 15 sec, 60°C for 10 sec, 72°C for 1 min). DNA cleanup was performed with 1.3 AMPure XP beads (Analis, Cat A63880) and DNA was eluted in 15 µl 0.1 TE buffer. The CUT&Tag libraries were analyzed on a Bioanalyzer (Agilent High Sensitivity DNA Chip, Cat 5067-4626) and sequenced on an Illumina NextSeq2000 instrument.  CUT&Tag