title: HCHO-K9me2-2
organism: Spodoptera frugiperda
description: Protocols: Sf9 cells are grown in F75 plates. They were seeded with 3E5 cells/mL/well. When confluence is reached, the medium was removed from the plates and cells were rinsed with cold 1X PBS. Cells were then scraped from the plate in 1mL 1X cold PBS. Cells were centrifuged for 2 minutes at 1500 rpm at 4°C. The supernatant was removed and the pellet resuspended in 300µl of TE buffer supplemented with Complete Mini Protease inhibitor cocktail. Cells were lyzed by sonication with a Diagenode Bioruptor (3 cycles, 30\" ON; 30\" OFF). Biological samples are being crushed in a douncer, in presence of 1% formaldehyde and incubated for 15 minutes to allow protein-DNA crosslinking. Crosslinking was quenched by the addition of 225mM glycin. Chromatin was then fragmented using a Bioruptor sonicator (Diagenode). An aliquot of sonicated chromatin at this stage is used as the Input sample. For the immunoprecipitation, sonicated chromatin is incubated for 4 h with the primary antibody at 4°C, then with Protein-A sepharose beads (CL4B) for again 4 h. Beads were then centrifuged and washed. Chromatin was eluted from the beads with a 50mM Tris-HCl buffer with 1%SDS and 10mM EDTA. To reverse the cross-linking, this immuno-precipitate was incubated overnight at 65°C and then 2-3h at 50°C with proteinase K. DNA was purified from this precipitate with 500µL of phenol-chloroform and 55µL of 4M LiCl. The DNA from the aqueous phase is precipitated with 100% ethanol and the pellet dried and resuspended in water. Chromatin was prepared from 50 L4 larvae pool and 50 mL of confluent cells per IP condition. Two biological replicates for input and ChIP conditions have been systematically produced except for larvae (1 replicate only). Library preparation were conducted by MGX GenomiX (IGF, Montpellier, France). Libraries were prepared with the TruSeq ChIP Sample preparation kit (Illumina). It contains 5 steps. 1-end-repair of DNA fragments. 2-Adenine addition at DNA ends. 3-Adapters ligation. 4-Adapters-ligated DNAs were then amplified by PCR. Only fragments containing two types of adapters are selected. 5-Purification on agarose gel 2%. Libraries were validated on Fragment Analyzer with a High Sensitivity NGS kit (Advanced Analytical Technologies, Inc) and quantified by qPCR with a Light Cycler 480 thermal cycler (Roche Molecular diagnostics).