An anterior part of the cerebral cortex of C57BL/6J mice submitted to a 6-h SD was sampled, and DNA was extracted using a DNeasy Blood & Tissue Kit (Qiagen). The remaining cerebral cortex was used for RNA extraction to perform qPCR validations (see above). Six pools of DNA (that is, three SD and three control) each including the DNA of three SD or three control mice (total nine mice per condition) were fragmented using a bioruptor (Diagenode, Denville, NJ, USA) and used for both methylation (5mC) and hydroxymethylation (5hmC) enrichments. 5 mC enrichment was performed as previously described.29 (link) 5hmC enrichment was performed using the Hydroxymethyl collector kit (ActiveMotif, Carlsbad, CA, USA). The DNA input and bound fractions were purified, amplified and labeled using Whole Genome Amplification (Sigma-Aldrich) and CGH Enzymatic Labeling (Agilent Technologies) kits.
Whole genome amplification
Manufactured by Merck Group
Whole genome amplification is a laboratory technique used to generate multiple copies of an entire genome from a small amount of DNA. It allows for the amplification of DNA samples, enabling further analysis and experimentation.
Automatically generated - may contain errors
3 protocols using whole genome amplification
1
Epigenetic Profiling of Mouse Cortex
2
Isolation and Analysis of Open Chromatin
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Disrupted or 'open' chromatin was isolated as described previously 48 . In brief, cell nuclei were digested with micrococcal nuclease and soluble chromatin released overnight followed by fractionation on a 6-40% isokinetic sucrose gradient in 80 mM NaCl, 0.1 mM EDTA, 0.1 mM EGTA and 250 μM PMSF. DNA purified from gradient fractions was analyzed by electrophoresis through 0.7% agarose in 1 × TPE buffer (90 mM Tris-phosphate, 2 mM EDTA) with buffer circulation. Preparative fractionation of DNA from gradient fractions was carried out by pulsed-field gel electrophoresis (PFGE) (CHEF system, Biorad) through 1% low melting point agarose in 0.5 × TBE, at 180 V, for 40 hr, with a 0.1-2 s switching time. Size markers were 1 kb (Promega) and λ-HindIII (NEB) DNA ladders. EtBr-stained gels were scanned using a 473 nm laser and a 580 nm band-pass filter on a Fuji FLA-3000. DNA of ~ 20 kb, corresponding to "open" chromatin, was isolated by β-agarase (NEB) digestion and amplified by whole genome amplification (Sigma) prior to microarray hybridization.
3
Isolation and Analysis of Open Chromatin
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Disrupted or 'open' chromatin was isolated as described previously 48 . In brief, cell nuclei were digested with micrococcal nuclease and soluble chromatin released overnight followed by fractionation on a 6-40% isokinetic sucrose gradient in 80 mM NaCl, 0.1 mM EDTA, 0.1 mM EGTA and 250 μM PMSF. DNA purified from gradient fractions was analyzed by electrophoresis through 0.7% agarose in 1 × TPE buffer (90 mM Tris-phosphate, 2 mM EDTA) with buffer circulation. Preparative fractionation of DNA from gradient fractions was carried out by pulsed-field gel electrophoresis (PFGE) (CHEF system, Biorad) through 1% low melting point agarose in 0.5 × TBE, at 180 V, for 40 hr, with a 0.1-2 s switching time. Size markers were 1 kb (Promega) and λ-HindIII (NEB) DNA ladders. EtBr-stained gels were scanned using a 473 nm laser and a 580 nm band-pass filter on a Fuji FLA-3000. DNA of ~ 20 kb, corresponding to "open" chromatin, was isolated by β-agarase (NEB) digestion and amplified by whole genome amplification (Sigma) prior to microarray hybridization.
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