title: GSM2055165: 4C_cortical astrocytes_digested; Mus musculus; OTHER
library_construction_protocol: Chromatin was prepared as described (Splinter et al. 2004) with minor modifications. Briefly, astrocytes (4 x 106) were pelleted by centrifugation (320g, 8 min, 4°C) and lysed (10 min) with ice cold lysis buffer (10 mM Tris pH 8.0, 10 mM NaCl, 0.2% NP-40, protease inhibitor cocktail-EDTA-free (Roche)). Cell nuclei were collected by centrifugation (600g, 5 min, 4°C). Nuclei were incubated with SDS (0.3% final conc., 1 hr, 37oC, with shaking). Triton-X-100 (1.8% final conc.) was added and the chromatin solution incubated (1 hr, 37oC, with shaking). Restriction buffer (NEB) was added (1x final concentration) before the chromatin was digested with MspI (C^CGG; 160U, New England Biolabs, 37oC, o/n, with shaking). MspI was inactivated (SDS [1.3% final conc.], 20 min, 65oC) and the chromatin solution diluted (10-fold) in water. Triton-X-100 (1% final conc.) was added and the digested chromatin incubated (1 hr, 37oC, with shaking) to sequester the unreacted SDS. A MspI digested E. coli amplicon (Supplementary Table S5 and Supplementary Fig. S1) was added at the same concentration as a diploid mouse locus, to control for random inter-molecular ligation events. T4 DNA ligase (40U, Invitrogen) was added and the chromatin ligated (4.5 hr, 16oC). Following ligation, crosslinks were reversed (3.56U Proteinase K (Fermentas), overnight, 65oC), before removal of RNA by incubation with DNase free RNAase A (0.06 mg, 45 min, 37oC). DNA was purified by three rounds of Chloroform:Phenol (1:1) extraction, addition of 12 µl LPA (10 mg/ml), 0.1x volume 3M Sodium Acetate and 2x volume absolute ethanol and precipitated (-20oC, 2 hr). DNA was pelleted by centrifugation (25 min, 13000rpm, RT) and subjected to two 70% ethanol washes (15 min, 13000rpm, RT). DNA was suspended in 100µl water A region spanning ~1/3 of the mtDNA genome (NC_010339, 3361-8410) encoding the ND1, ND2, CO1, CO2, ATPase8, ATPase 6 genes was digested in silico with MspI to produce five 4C baits. Inverted PCR primer pairs (Supplementary Table S4) were designed (Primer3 (v. 0.4.0);(Untergasser et al. 2012; Koressaar & Remm 2007)) to amplify out of each individual bait. Blocker primers (Karkare & Bhatnagar 2006; Vestheim & Jarman 2008) were designed (Supplementary Table S5) to hybridise to the adjacent baits. Blocker primers were modified with three carbons on their 5’ and 3’ ends to prevent amplification of the sequences to which they anneal by blocking polymerase activity. PCR reactions contained bait-specific combinations of primers and blockers, and KAPA long range polymerase (Supplementary Table S6). PCR reaction conditions for baits 1, 3, 4, and 5 were (94 °C, 3:00 min; 35x [94°C, 0:25 sec; 55°C, 0:15 sec; 72°C, 5:00 min]; 72°C, 7:00min). PCR conditions for bait 2 were (94°C, 3:00 min; 35x [94°C, 0:25 sec; 41°C, 0:15 sec; 72°C, 5:00 min]; 72°C, 7:00min). Four 20µl PCR reactions were completed for each of the 4C baits. PCR products (10µl) were visualized following separation on a 1.5% agarose gel (Supplementary Fig. S3). Following amplification, PCR products (M1 to M5 for each replicate) were pooled and column purified (QIAquick PCR Purification Kit). Purified libraries (cut library, 3µg; CL1 & 2 libraries, 6µg) were sequenced on a HiSeq 2000 (Beijing Genomics Institute, Hong Kong. Between 29 and 56 million paired-end 100 bp sequences were obtained for each library
attributes.tag: GEO Accession
attributes.value: GSM2055165