GSM7612255: astrocytes, p65, DMSO, inflamm, ChIP, biol rep 1; Mus musculus; ChIP-Seq
3–4 million astrocytes were fixed in 1% formaldehyde (F1635, Sigma). Fixation was quenched by the addition of glycine-PBS (Nacalai Tesque) at a final concentration of 0.125 M, followed by nuclear membrane extraction using Farnham lysis buffer (5 mM PIPES, pH 8.0; 85 mM KCl; 0.5% NP-40) supplemented with a protease inhibitor cocktail (Nacalai Tesque).  Pellets of extracted membrane were resuspended in RIPA buffer (10 mM Tris-HCl, pH 7.4; 1 mM EDTA, pH 8.0; 0.1% SDS; 0.1% sodium deoxycholate; 1% Triton X-100). Sonication was performed using the Branson Sonifier (SFX-550) at 35% amplitude, using 12 cycles of 20 s sonication and 30 s of pause at 4°C. Chromatin was clarified using centrifugation at maximum speed and pre-cleared with 40 μL of washed Dynabeads protein A (ThermoFisher) for 30 min at 4°C, followed by immunoprecipitation using 40 μL of washed Dynabeads protein A and 5 μL of control IgG, anti-p65 p65 (CST, #8242), or anti-Nrf2 antibodies NRF2 (CST, #12721). After overnight incubation at 4°C, the beads were washed twice with cold RIPA buffer, twice with RIPA buffer containing 0.3 M NaCl, twice with LiCl buffer (0.25 M LiCl; 0.5% Igepal-630; 0.5% sodium deoxycholate), once with TE (10 mM Tris, pH 8.0; 1 mM EDTA) and 0.2% Triton X-100, and once with TE. Cross linking was reversed by incubating the beads at 65°C for 4 h and incubated at 37°C for 30 min with 0.5 μg of RNaseA (ThermoFisher), then at 50°C for 1 h in the presence of 0.3% SDS and 1 mg/mL Proteinase K (Nacalai Tesque). DNA was purified using Zymo ChIP DNA clean and Concentrator kit (Zymo Research). Total ChIP DNA was used to prepare Illumina sequencing libraries. End-repair was performed in 75 μL of T4 ligase reaction buffer, 0.4 mM dNTPs, 4 U of T4 DNA polymerase (NEB), 13.5 U of T4 Polynucleotide Kinase (NEB), and 1.5 U of Klenow fragment (NEB) at 24°C for 30 min in a ThermoMixer C (Eppendorf, Hamburg, Germany) at 400 rpm. End-repair reaction was cleaned using 2x Agencourt AMPure XP beads (Beckman Coulter, CA, USA) and eluted in 15 μL of EB. The eluate was used for the A-tailing reaction in 30 μL of NEBNext dA-Tailing reaction buffer (NEB) with 7.5 U of Klenow fragment exo- (NEB) at 37°C for 30 min. Thirty microlitres of the A-tailing reaction mixture was mixed with Quick Ligase Buffer 2X (NEB), 3,000 U of Quick ligase, and 5 nM annealed adaptor (Illumina truncated adaptor), and the total volume was made up to 75 μL; the mixture was incubated at 25°C for 20 min. Adaptor was prepared by annealing the following HPLC-purified oligos: 5′-phosphate/GATCGGAAGAGCACACGTCT-3′ and 5′-ACACTCTTTCCCTACACGACGCT CTTCCGATC∗T-3′ (∗phosphorothioate bond). Ligation was stopped by adding 50 mM EDTA and cleaned with 1.8x Agencourt AM- Pure XP beads and eluted in 15 μL of EB. The eluate was used for PCR amplification in a 50 μL reaction mixture with 1 μM primers (TruSeq barcoded primer p5, AATGATACGGCGACCACCGAGATCTACACNNNNNNNNACACTCTTTCCCTACACGACGCTCTTCCGATC∗T; TruSeq barcoded primer p7, CAAGCAGAAGACGGCATACGAGANNNNNNNGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC∗T; NNNNNNNN represents barcode and ∗ represents a phosphothiorate bond), and 2X Kapa HiFi HotStart Ready mix (Kapa Biosystems, MA, USA). The PCR conditions were as follows: 45 s at 98°C followed by 15 cycles of 15 s at 98°C, 30 s at 63°C, and 30 s at 72°C, and a final 5-min extension at 72°C. PCR products were cleaned with Agencourt AMPure XP beads (Beckman Coulter), run on a 2% agarose gel, and a smear of gel corresponding to 200–500 bp DNA in size was cut and gel purified using QIAquick Gel Extraction Kit (QIAGEN). Library concentration was determined using KAPA Library Quantification Kit for Illumina Platforms (Kapa Biosystems). Sequencing was performed on Illumina Nextseq500 (75-bp single-end reads).