GSM5692878: craniofacial tissues - H3K4me3_rep1; Sminthopsis crassicaudata; ChIP-Seq
Chromatin immunoprecipitation was performed using the MAGnify Chromatin Immunoprecipitation System (Thermo Fisher, 492024) according to manufacturer's instructions. Briefly, frozen dunnart tissue samples were diced quickly with two razor blades in cold dPBS (Gibco) followed by crosslinking in 1\% formaldehyde solution (Sigma) for 10 minutes. I then added 0.125 M glycine and incubated for 5 minutes at room temperature to neutralize the formaldehyde. Chromatin was fragmented to 300bp average size by sonication on a Covaris S2 using the following parameters: duty cycle = 5%, intensity = 2, cycles per burst = 200, cycle time = 60 seconds, cycles = 10, temperature = 4°C, power mode = frequency sweeping, degassing mode = continuous. In each ChIP experiment, I used sheared chromatin from each replicate for immunoprecipitation with antibodies against H3K4me3 (abcam ab8580) and H3K27ac (abcam ab4729). An input control was included for each replicate. DNA was purified according to kit instructions using a DynaMag PCR Magnet (Thermo Fisher). Illumina sequencing libraries were prepared from ChIP-enriched DNA by GENEWIZ (Suzhou, China). Libraries were constructed following the manufacturer's protocol (NEBNext UltraTMII DNA Library Prep Kit for Illumina). For each sample, a minimum of 10 ng of ChIP product was used and libraries were multiplexed and sequenced on an Illumina HiSeq instrument according to manufacturers instructions (Illumina, San Diego, CA, USA). Sequencing was carried out using a 2x150 paired-end configuration.