title: Illumina HiSeq 2500 paired end sequencing; 5-hydroxymethylcytosine (5hmC) methylation profiling of adult mouse brain and liver tissues (hMeDIP-seq)
design: 5-hydroxymethylcytosine (5hmC) methylation profiling of adult mouse brain and liver tissues (hMeDIP-seq)
library_construction_protocol: Wild-type male and female mice at 3 months of age were purchased from National Laboratory Animal Center and maintained in pathogen-free facility. Mice were sacrificed with five brain sub-regions (cortex, hippocampus, thalamus, hypothalamus, and cerebellum) and liver being harvested. The animal protocol was approved by the Institutional Animal Care and Use Committee of the Taipei Medical University. Purified genomic DNA was sonicated into short fragments with a size range of 100-500 bp by a Bioruptor (Diagenode, Sparta, NJ). The sheared DNA was then subjected to enrich 5-hmC-containing DNA using the Hydorxymethyl Collector kit (Active Motif, Carlsbad, CA) according to manufacturer's instruction. The resulting enriched 5-hmC DNA was then subjected to sequencing library construction. Tissues were lysed in the NTES buffer [1 M NaCl, 0.5% SDS, 10 mM Tris-HCl (pH 8.0), 1 mM EDTA] with proteinase K (200 μg/ml). Total DNA was extracted twice with phenol-chloroform, precipitated with ethanol, and finally dissolved in autoclaved water. 10 nanogram of enriched 5-hmC DNA and sheared genomic DNA without 5-hmC enrichment were end-repaired, A-tailed, and ligated to adaptors using Illumina TruSeq ChIP Sample Preparation kit (Illumina). The adapter-ligated 5-hmC library was amplified by PCR for 10 cycles using KAPA HiFi DNA polymerase (Kapa Biosystems).
attributes.tag: Experimental Factor: hippocampus
attributes.value: organism part
attributes.tag: Experimental Factor: male
attributes.value: sex
attributes.tag: Experimental Factor: 5hmC enrichment (Active Motif)
attributes.value: treatment