title: GSM2756643: s2 cell H3K4me2 ChIP-seq rep1; Drosophila melanogaster; ChIP-Seq
library_construction_protocol: For H3K4me2 ChIP-seq, formaldehyde was added to media to a final concentration of 1% and incubated for 10 minutes on a shaker at room temperature. The reaction was quenched by adding glycine to a final concentration of 125 mM followed by 5 minutes of incubation on a shaker at room temperature. The cells were collected by centrifugation at 500 x g for 5 min at 4 °C and washed twice with ice-cold PBS. The cell pellet was resuspended in 10 ml ice-cold cell lysis buffer (5 mM HEPES (pH8), 85 mM KCl, 0.5% NP-40) with protease inhibitors (cOmplete™ ULTRA Tablets, Mini, EDTA-free, EASYpack Protease Inhibitor Cocktail, Roche # 05892791001) and incubated for 10 minutes at 4°C. Nuclei were released by 10 strokes with a Wheaton Dounce Homogenizer (tight pestle). The crude nuclear extract was collected by centrifugation at 500 x g for 5 min at 4°C, resuspended in 1 ml ice–cold nuclear lysis buffer (50 mM HEPES (pH 8), 10 mM EDTA, 0.5% N-Lauroylsarcosine with protease inhibitors) and incubated for 20 minutes at 4°C. After addition of 1 ml nuclear lysis buffer samples were sonicated using a Diagenode Bioruptor for 18 cycles (30” ON / 30” OFF) on “high”. After sonication samples were centrifuged at 14000 x g for 10 minutes at 4°C and the supernatant was aliquoted to DNA-low binding tubes. The chromatin was flash frozen in liquid nitrogen and stored at -80°C. Protein A Sepharose (PAS) beads (Sigma #P9424) were washed twice with RIPA140 (140 mM NaCl, 10 mM Tris-HCl (pH 8), 1mM EDTA, 1% Triton X100, 0.1% SDS, 0.1% Na-Deoxycholate) with proteinase inhibitors and 1mg/ml BSA (Sigma #A7906) and incubated overnight. Chromatin was thawed on ice. 50µg of Chromatin was used per ChIP experiment. RIPA140 with proteinase inhibitor was added to a total volume of 1 ml and incubated with 2 µg of H3K4me2 antibody (Abcam, ab32356, Lot#: GR209821-1; or Epicypher 13-0013, Lot#: 14247001) for 16 hours at 4°C on a rotating mixer (40 rpm). 1% of the chromatin was used as input controls. Blocked beads were added to the chromatin-antibody complex solution and incubated for 3 hours at 4°C on a rotating mixer at 40 rpm. Complexes were washed once with 1ml RIPA140, 4 times with 1 ml RIPA500 (500 mM NaCl, 10 mM Tris-HCl (pH 8), 1mM EDTA, 1% Triton X100, 0.1% SDS, 0.1% Na-Deoxycholate) for 10 minutes each. Complexes were subsequently washed once in 1 ml LiCl-Buffer (250 mM LiCl, 10 mM Tris-HCl (pH 8), 1mM EDTA, 0.5% NP-40, 0.5% Na-Deoxycholate) and TE (10mM Tris-HCl (ph 8), 1mM EDTA) for 2 minutes each. Between each wash, beads were spun down at 500 x g for 2 minutes and the supernatant was discarded. Beads were resuspended in 100 µl TE and RNase A was added to a final concentration of 50 µg/ml followed by incubation at 37°C for 30 minutes. The samples were adjusted to a final concentration of 0.5% SDS and Proteinase K was added to a final concentration of 500µg/ml. Proteins were digested at 37°C for 90 minutes followed by reverse cross-linking overnight at 65°C. DNA was purified using phenol-chloroform extraction followed by ethanol precipitation. Libraries were prepared using the NEXTflex qRNA-Seq Kit v2 from Bioo Scientific (Catalog #5130-11) and sequenced on an Illumina NextSeq 500 sequencer.
attributes.tag: GEO Accession
attributes.value: GSM2756643