title: GSM2100392: H3K4me3_Adult_Rep1; Amphimedon queenslandica; ChIP-Seq
library_construction_protocol: Approximately a cubic centimeter of adult sponge tissue was squeezed through a fine cloth and cells were crosslinked in 2% formaldehyde for 5 min at room temperature (RT). Larvae (~350) were pooled, homogenized and crosslinked as above. A similar procedure was then adopted for both developmental stages. Crosslinking was quenched with 125 mM glycine for 5 min at RT. Cells were washed twice in filtered seawater and centrifuged at 500g for 5 min. Pelleted cells were lysed in SDS Lysis buffer (10 mM EDTA, 50 mM Tris-HCl at pH 8.0, 1% SDS, plus protease and phosphatase inhibitors), incubated for at least 10 minutes on ice, and sonicated for 12 min (12 cycles, each one 30sec “ON”, 30sec “OFF”) in a Bioruptor Sonicator (Diagenode, Seraing, Belgium) in order to generate 200-300 bp fragments. Optimal sonication conditions were previously set up by testing a range of sonication cycles (from 5 to 30), determining that 12 cycles were the optimal. Non-soluble material was removed from the lysate by centrifugation at 12,000g for 10 min at 4°C. An aliquot of the soluble material was removed for input DNA and stored at -20°C. To reduce the SDS concentration to 0.1%, the remaining soluble material was diluted 10 fold in ChIP dilution buffer (1.2 mM EDTA, 16.7 mM Tris-HCl at pH 8.0, 167 mM NaCl, 1.1% Triton X-100, 0.01% SDS, plus protease and phosphatase inhibitors). To reduce non-specific background, the diluted soluble material was pre-cleared with Dynabeads protein G beads (#10003D, ThermoFisher, Waltham, MA), and, at the same time, the antibodies were linked to Dynabeads protein G beads (#10003D, ThermoFisher), rotating for one hour at 4°C. At this point, the pre-cleared diluted soluble material was incubated with the antibody-bead mixtures, rotating at 4°C overnight. Immunoprecipitated material was washed three times with Low Salt Wash Buffer (2 mM EDTA, 20 mM Tris-HCl at pH 8.0, 150 mM NaCl, 1% Triton X-100, 0.1% SDS), three times with High Salt Wash Buffer (2 mM EDTA, 20 mM Tris-HCl at pH 8.0, 500 mM NaCl, 1% Triton X-100, 0.1% SDS), three times with LiCl Wash Buffer (1 mM EDTA, 1 mM Tris-HCl at pH 8.0, 1% DOC, 1% NP-40, 250 mM LiCl), and three times with TE buffer. DNA complexes were eluted 30 min at 65°C with TE-SDS (1X) and decrosslinked overnight at 65°C, along with input DNA, with the addition of 125 mM NaCl. Decrosslinked DNA complexes and input DNA were treated with RnaseA, and subsequently with proteinase K. Finally, immunoprecipitated and input DNA were purified with phenol:chloroform:isoamyl extraction (25:24:1), recovered by precipitation with ethanol in the presence of 300 mM NaOAc pH 5.2 and 2 μl of glycogen carrier (10 mg/ml), and resuspended in UltraPure™ DNase/RNase-Free Distilled Water (ThermoFisher) for later use. Libraries of immunoprecipitated DNA and input DNA were prepared using the NEBNext ChIP-seq Library Prep Master Mix Set for Illumina (#E6240, New England Biolabs, Ipswich, MA) according to the manufacturer's protocol. The quality and profile of the libraries was analyzed using Agilent High Sensitivity DNA Kit (#5067-4626, Agilent, Santa Clara, CA) and quantified using KAPA Library Quantification Kits (#KK4824, Kapa Biosystems, Wilmington, MA). Deep sequencing (paired-end 100bp) of the adult libraries – two biological replicates for H3K4me3, H3K4me1, H3K36me3, H3K27me3, RNAPII, input DNA and no biological replicates for H3K27ac and total histone H3 – was performed by the Macrogen Oceania NGS Unit on Illumina HiSeq 2000 instrument (Illumina, San Diego, CA, United States). Deep sequencing (paired-end 40bp) of the larva libraries – no biological replicates for H3K4me3, H3K4me1, H3K27me3, H3K27ac, RNAPII, input DNA – was performed by the Central Analytical Research facility (CARF), Brisbane, Queensland, Australia, on Illumina NextSeq 500 instrument (Illumina, San Diego, CA, United States).
attributes.tag: GEO Accession
attributes.value: GSM2100392