title: GSM4293955: mESC_Pde10a_17; Mus musculus; OTHER
library_construction_protocol: Striatal tissue: Frozen striata from 4 mice were homogenized in ice-cold PBS supplemented with 1x Protease Inhibitors Cocktail (PIC, cOmplete EDTA free, Roche) and cross-linked in 2% formaldehyde for 15 minutes at room temperature. Cross-linking was stopped by the addition of glycine to final concentration 0.125 M and tissue was washed using ice-cold PBS. Cells were lysed then in Cell Lysis Buffer (10mM Hepes pH8; 85mM KCl; 0.5% NP-40) and nuclei were were collected after treatment with Nuclear Extraction Buffer (0.5% SDS, 10mM EDTA pH8, 50mM Tris). Nuclei were counted and aliquoted at 5 million nuclei/tube. 5 million nuclei aliquots were further permeabilized by treatment for 1 h with 0.4% SDS at 37°C, then a further 1h with 2.6% Triton-X100 at 37°C. Nuclei were digested overnight with 1000 U DpnII at 37°C, then washed twice by centrifuging and resuspending in T4 DNA ligase buffer. In situ ligation was performed in 400 μL T4 DNA ligase buffer with 20,000 U T4 DNA ligase overnight at 16°C. DNA was purified by reverse cross-linking with an overnight incubation at 65°C with proteinase K, followed by RNase A digestion, phenol/chloroform extraction and isopropanol precipitation. The DNA was digested with 5 U/μg Csp6I at 37°C overnight, then re-purified by phenol/chloroform extraction and isopropanol precipitation. The DNA was then circularized by ligation with 200 U/μg T4 DNA ligase under dilute conditions (5 ng/μL DNA), and purified by phenol/chloroform extraction and isopropanol precipitation. mESC: Cells were detached with trypsin, washed by centrifugation in PBS, and then fixed with 2% formaldehyde in mES culture medium for 10 min at 23°C. The fixation was quenched with cold glycine at a final concentration of 125 mM, then cells were washed with PBS and permeabilized on ice for 1 h with 10 mM Tris-HCl, pH 8, 100 mM NaCl, 0.1% NP-40 and protease inhibitors. Nuclei were resuspended in DpnII restriction buffer at 10 million nuclei/mL concentration, and 5 million nuclei aliquots were further permeabilized by treatment for 1 h with 0.4% SDS at 37°C, then a further 1h with 2.6% Triton-X100 at 37°C. Nuclei were digested overnight with 1000 U DpnII at 37°C, then washed twice by centrifuging and resuspending in T4 DNA ligase buffer. In situ ligation was performed in 400 μL T4 DNA ligase buffer with 20,000 U T4 DNA ligase overnight at 16°C. DNA was purified by reverse cross-linking with an overnight incubation at 65°C with proteinase K, followed by RNase A digestion, phenol/chloroform extraction and isopropanol precipitation. The DNA was digested with 5 U/μg Csp6I at 37°C overnight, then re-purified by phenol/chloroform extraction and isopropanol precipitation. The DNA was then circularized by ligation with 200 U/μg T4 DNA ligase under dilute conditions (5 ng/μL DNA), and purified by phenol/chloroform extraction and isopropanol precipitation. 50 ng aliquots of 4C DNA were used as template for PCR with bait-specific primers containing Illumina adapter termini (primer sequences and optimal PCR conditions available on request). PCR reactions were pooled, primers removed by washing with 1.8x AMPure XP beads, then quantified on a Bioanalyzer (Agilent) before sequencing with a HiSeq 4000 (Illumina).
attributes.tag: GEO Accession
attributes.value: GSM4293955