title: GSM6426703: H3K36me3 ChIP-seq 2; Aedes aegypti; ChIP-Seq
library_construction_protocol: Aag2 cells were cross-linked with 1% formaldehyde for 10 min at room temperature with gentle shaking. Cross-linking was quenched with the addition of 1/10 volume 1.25 M glycine. Cells were washed with 1× PBS, then harvested by scraping in Buffer B (20 mM HEPES, 0.25% Triton X-100, 10 mM EDTA, and 0.5 mM EGTA). Cells were then pelleted by spinning at 600 × g for 5 min at 4 °C. Cell pellet was resuspended in Buffer C (150 mM NaCl, 50 mM HEPES, 1 mM EDTA, and 0.5 mM EGTA) and rotated for 10 min at 4 °C. Cells were again pelleted by spinning at 600 × g for 5 min at 4 °C. The cell pellet was resuspended in 1× incubation buffer (0.15% SDS, 1% Triton X-100, 150 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, and 20 mM HEPES) at 15 million cells/mL. Cells were sheared with a Bioruptor Pico sonicator (Diagenode) at 4 °C using 6 cycles of 30 s on, 30 s off. Sonicated chromatin was spun at 18,000 × g for 5 min at 4 °C. Chromatin in the upper supernatant was aliquoted, immediately frozen in liquid nitrogen, and stored in -80°C.  Sonicated chromatin from 2 million cells were used for each ChIP experiment. Chromatin was incubated in 1× incubation buffer (0.15% SDS, 1% Triton X-100, 150 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, and 20 mM HEPES) supplemented with 1× protease inhibitors, 0.1% BSA, and a 50:50 mix of Protein A and G Dynabeads (Invitrogen), with agitation overnight at 4 °C. Generally, 1 µg antibody was used for each ChIP (H3K4me3, Diagenode, C15410003; H3K27ac, Diagenode, C15410196; H3K36me3, Diagenode, C15410058; H3K9me3, Diagenode, C15410193; RNA pol II, Santa cruz, sc-56767; RNA pol II ser5p, Abcam, ab5131; RNA pol II ser2p, Chromotek, 3E10). As a control, no antibody was added to ChIP input sample (sonicated chromatin). The beads were washed twice with wash buffer 1 (0.1% SDS, 0.1% sodium deoxycholate, 1% Triton X-100, 150 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, and 20 mM HEPES), once with wash buffer 2 (wash buffer 1 with 500 mM NaCl), once with wash buffer 3 (250 mM LiCl, 0.5% sodium deoxycholate, 0.5% NP-50, 1 mM EDTA, 0.5 mM EGTA, and 20 mM HEPES), and twice with wash buffer 4 (1 mM EDTA, 0.5 mM EGTA, and 20 mM HEPES). After the washing steps, beads were rotated for 20 min at room temperature in elution buffer (1% SDS and 0.1 M NaHCO3). The supernatant was de-cross-linked with 200 mM NaCl and 100 μg/mL proteinase K for 4 h at 65 °C. De-cross-linked DNA was purified with MinElute PCR Purification columns (Qiagen). DNA concentrations were determined by Qubit fluorometric quantification (ThermoFisher Scientific). Libraries were prepared with the Kapa Hyper Prep Kit for Illumina sequencing (Kapa Biosystems) according to the manufacturer's protocol with the following modifications. 3 ng DNA was used as input, with NEXTflex adapters (Bioo Scientific) and ten cycles of PCR amplification. Post-amplification cleanup and size selection was performed with dual AMPure XP beads selection (0.6× and 0.8×). Size-selected samples were analyzed for purity with a High Sensitivity DNA Chip on a Bioanalyzer 2100 system (Agilent), with an average size between 300 and 500 bp.