GSM4859751: Female_H3K36me3_Rep2; Ectocarpus sp.; ChIP-Seq
For ChIP-seq, Ectocarpus tissue was fixed for five minutes in seawater containing 1% formaldehyde and the formaldehyde eliminated by rapid filtering followed by incubation in PBS containing 400 mM glycine. Nuclei were isolated by grinding in liquid nitrogen and in a Tenbroeck Potter in nuclei isolation buffer (0.1% triton X-100, 125 mM sorbitol, 20 mM potassium citrate, 30 mM MgCl2, 5 mM EDTA, 5 mM β-mercaptoethanol 55 mM HEPES at pH 7.5 with complete ultra protease inhibitors), filtering through Miracloth and then washing the precipitated nuclei in nuclei isolation buffer with and then without 1% triton X-100. Chromatin was fragmented by sonicating the purified nuclei in nuclei lysis buffer (10 mM EDTA, 1% SDS, 50 mM Tris-HCl at pH 8 with complete ultra protease inhibitors) in a Covaris M220 Focused-ultrasonicator (duty 25%, peak power 75, cycles/burst 200, duration 900 sec at 6°C). The chromatin was incubated with an anti-histone PTM antibody overnight at 4°C and the immunoprecipitation carried out using Dynabeads protein A and Dynabeads protein G. Following immunoprecipitation and washing, a reverse cross-linking step was carried out by incubating for at least six hours at 65°C in 200mM NaCl and the samples were then digested with Proteinase K and RNAse A. The purified DNA was then prepared for Illumina sequencing. For RNA-seq, RNA was extracted from the same duplicate male and female gametophytes cultures as were used for the ChIP-seq analysis using the Qiagen RNeasy plant mini kit with an on-column Deoxyribonuclease I treatment. RNA concentration have been assessed using a Qubit 2.0 Fluorometer (Thermofisher). RNA contamination for genomic DNA have been tested by PCR using pairs of primers specific to the non-coding genome. Illumina librairies were prepared by Fasteris using the TruSeq ChIP-seq (for ChIP-seq) or TruSeq RNA stranded protocol (for RNA-seq) before sequencing.