title: GSM9003709: Input in TKO rep2; Mus musculus; ChIP-Seq
library_name: GSM9003709
library_construction_protocol: ChIP DNA was extracted by Native ChIP protocol At least 2 × 105 cells were lysed in 50 µl Buffer 1 (60 mM KCl, 15 mM NaCl, 5 mM MgCl2, 0.1 mM EGTA, 15 mM Tris-HCl (pH7.5), 0.3 M Sucrose, 0.5 mM DTT, and protease inhibitors (Nacalai tesque, 04080-11), then were added 50 µl Buffer2 (Buffer 1 + 1% NP40) to the sample. After the incubation for 10 min on ice, the sample was added 800 µl Buffer 3 (60 mM KCl, 15 mM NaCl, 5 mM MgCl2, 0.1 mM EGTA, 15 mM Tris-HCl (pH7.5), 1.2 M Sucrose, 0.5 mM DTT, and protease inhibitors). After centrifugation at 9000 rpm for 10 min at 4°C, the sample was added 100 µl MNase buffer (0.32 M Sucrose, 50 mM Tris-HCl (pH7.5), 4 mM MgCl2, 1 mM CaCl2, and phenylmethylsulfonyl fluoride (PMSF)) followed by incubation for 20 min at 37 °C with 0.3 U MNase (TAKARA, 2910 A). After the incubation, the reaction was stopped by 10 µl 0.5 M EDTA. After centrifugation at 15,000 rpm for 10 min at 4°C, the supernatant was added 900 µl Incubation buffer (50 mM NaCl, 20 mM Tris-HCl (pH7.5), 5 mM EDTA, 0.01% NP40, ad PMSF). In all, 10% of the sample was used for input, and the remains proceeded the following procedure. The sample was mixed with the antibody-beads complex which was formed by incubation of 20 µl protein A/G beads (Santa Cruz, sc-2003) with antibody in Incubation buffer on ice for 1 h. After rotating for overnight at 4°C, the complex was washed by 500 µl Wash buffer A (75 mM NaCl, 50 mM Tris-HCl (pH7.5), 10 mM EDTA, and 0.01% NP40), 500 µl Wash buffer B (100 mM NaCl, 50 mM Tris-HCl (pH7.5), 10 mM EDTA, and 0.01% NP40), and 500 µl wash buffer C (175 mM, 50 mM Tris-HCl (pH7.5), 10 mM EDTA, and 0.01% NP40). The ChIP DNA was eluted by RNase and Protease treatment followed by DNA purification using PCR purification kit (QIAGEN).
identifiers.namespace: GEO
identifiers.id: GSM9003709_r1