title: GSM4548670: WT input repl. 2; Caenorhabditis elegans; ChIP-Seq
library_construction_protocol: Synchronized L3 larvae were washed off the plates using isotonic M9 solution. Fixation was performed for 30 min at 20°C in crosslinking solution (2% paraformaldehyde in M9 buffer), and excess formaldehyde was quenched with 0.1 M Tris-HCl pH 7.5. The pellet was then washed twice with M9 buffer at 4°C. The worm pellet was resuspended in 1 mL ice-cold RIPA buffer (10 mM Tris-HCl pH 8.0, 0.1% SDS, 1% Triton X-100, 0.1% sodium deoxycholate, 1 mM EDTA, 0.15 M NaCl) supplemented with Halt™ Protease Inhibitor Cocktail (Thermo Fisher Scientific). The suspension was then transferred to a Covaris milliTUBE 1 mL with AFA® fiber and shearing was performed using the following settings in SonoLab7: Peak Incident Power (PIP) = 240W; Duty Factor = 20%; Cycles per burst (cpb) = 200; time = 480 seconds. After shearing, the crude extract was spun for 10 min at 10,000 x g and the supernatant was collected. Protein concentration was determined by the Bradford method. Extract corresponding to 1 mg protein was diluted to 500 μl with ice-cold RIPA buffer supplemented with Halt™ Protease Inhibitor Cocktail (Thermo Fisher Scientific). Antibody specific for H3K9me2 (Abcam, ab1220, 5 μg) was added and, after incubation at 4°C for 1 h, 50 μl of Dynabeads™ Protein G suspension (Thermo Fisher Scientific) were added and the mixture was incubated at 4°C for 1 h on rotation. Beads were recovered with a Dynal® Magnetic Particle Concentrator (Invitrogen) and washed at 4°C with: 0.5 mL buffer TSE-150 (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.1, 150 mM NaCl), 2 X 0.5 mL buffer TSE-500 (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.1, 500 mM NaCl), 0.5 mL buffer TSE-1M (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.1, 1 M NaCl), 0.5 mL buffer LiCl (250 mM LiCl, 1% NP40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8.1), 2 X 0.5 mL buffer TE (10 mM Tris pH 8.0, 1 mM EDTA). Antibody-bound chromatin was recovered in 2 X 0.2 mL elution buffer (1% SDS, 0.1 M NaHCO3) by shaking at room temperature for 15 min. Elution buffer was added to input samples (5%) to a total volume of 0.4 mL. Input and ChIP chromatin samples were subsequently processed in parallel. After 20 μl of 5 M NaCl was added to each sample, cross-links were reversed overnight at 65°C. 10 μl of 0.5 M EDTA, 20 μl of 1 M Tris-HCl pH 6.5 and 0.5 μl of 20 mg/mL Proteinase K (Thermo Fisher Scientific) were added and each sample was incubated for 1 h at 42°C. DNA was recovered using the QIAquick PCR Purification Kit (Qiagen) in accordance with the manufacturer's instructions. Immunoprecipitated DNA and input libraries were prepared using TruSeq ChIP Library Preparation kit following the manufacturer's instructions (Illumina) and checked for concentration and quality on DNA chips with a Bioanalyzer (Agilent). 75-bp single read sequences were generated on the NextSeq 500 sequencer according to manufacturer's instructions (Illumina).
attributes.tag: GEO Accession
attributes.value: GSM4548670