GSM7124208: IgG_r_Cortex; Mus musculus; ChIP-Seq
Adult neocortical tissue was acutely isolated from two-month-old C57BL/6J mice (n=3 males). To do so, animals were deeply anesthetized with an intraperitoneal injection of a mixture of tiletamine and zolazepam (40 mg/kg; Zoletil; Virbac) and xylazine (5 mg/kg; Rompun; Bayer) and the brain removed after rapid decapitation. Neocortices from both hemispheres were microdissected and pooled together, frozen and stored at -80°C until further analysis. Around 10 mg of neural tissue were chopped with a scalpel, harvested in 5 ml of 0.01 M PBS (pH 7.4) and crosslinked by adding formaldehyde to a final concentration of 1.5%, incubated for 30 min at RT on a rotator, and quenched with 0.125 M glycine for 5 min at RT. After crosslinking, the tissue was washed twice in cold 0.01 M PBS (pH 7.4) and centrifuged at 1.000xg for 5 min at 4°C. Tissue pellet was resuspended in 0.25ml of sodium dodecyl sulfate (SDS) lysis buffer (50 mM Tris pH 8.0, 1% SDS, 10 mM EDTA, anti-proteases) and incubated on a rotator for 30 min at 4°C. Afterwards, sample was sonicated for 18 cycles on high power setting (30s ON, 30s OFF) using the Bioruptor Next Gen (Diagenode) and then centrifuged at 20.000xg for 10 min at 4°C.  The isolated chromatin was diluted 10-fold with ChIP dilution buffer (16.7 mM Tris-HCl pH 8.0, 0.01% SDS, 1.1 % Triton X-100, 1.2 mM EDTA, 167 mM NaCl) (1/10 was kept as input) and incubated with 2 µg of antibody (rabbit anti-COUP-TFI/Nr2f1; Studer's lab) overnight at 4°C on a rotator. Protein G-conjugated magnetic beads (Dynal, Thermo Fisher Scientific) were saturated with PBS/1% BSA overnight at 4°C. Next day, samples were incubated with saturated beads for two hours at 4°C on a rotator, and subsequently washed with 1 ml of cold Low salt buffer (20 mM Tris-HCl pH 8.0, 0.1 % SDS, 1% Triton X-100, 2 mM EDTA, 150 mM NaCl), 1 ml of cold High salt buffer (20 mM Tris-HCl pH 8.0, 0.1 % SDS, 1% Triton X-100, 2 mM EDTA, 500 mM NaCl), 1 ml of cold LiCl buffer (10 mM Tris-HCl pH 8.0, 1% DOC, 250 mM LiCl, 1 mM EDTA, 1% NP-40), and twice with 1 ml of cold TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA). The immunoprecipitated chromatin was eluted with 200 µl of Elution buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 1% SDS, 150 mM NaCl, 5mM DTT) for 30 min at RT on a rotator, and decrosslinked at 65°C overnight. The decrosslinked DNA was purified using QIAquick PCR Purification Kit (Qiagen) according to the manufacture's instruction. sequencing libraries were constructed using the NEBNext ChIP-seq Library Prep Reagent Set for Illumina