GSM2500899: Tg197 11weeks MethylCap-Seq replicate 2; Mus musculus; MBD-Seq
RNA was extracted from mouse SFs using the Absolutely RNA Miniprep Kit (Agilent Technologies) and from human SFs using the miRNeasy Mini kit (Qiagen). DNA was extracted from mouse SFs using the PureLink Genomic DNA Mini Kit (Invitrogen). In order to enrich for methylated DNA, the MethylMiner DNA Enrichment Kit from Invitrogen was used as per the manufacturer’s instructions. DNA was firstly fragmented by sonication to an average size of 250 bp. Then enrichment was performed with a double elution (high and low salt) in order to capture both sparsely- and densely-methylated DNA; fractions were combined in the end. To enrich for H3K4me3, DNA was processed using the iDeal ChIP-seq Kit (Diagenode) in combination with a ChIP-grade H3K4me3 antibody (Diagenode), as per the manufacturer’s instructions. Chromatin was firstly sheared by sonication using a Bioruptor (Diagenode) and then subjected to the H3K4me3 enrichment protocol with a minor modification: after performing an RNase I digestion for 30 min at 37oC, the de-crosslinked DNA was eluted using the QIAquick PCR Purification Kit (Qiagen). Enrichment (in comparison to a non-enriched input control) was confirmed by quantitative PCR. All library preparations, next generation sequencing and quality control steps were performed by the McGill University and Genome Quebec Innovation Centre. More specifically, for RNA-seq, TruSeq RNA libraries were prepared and samples were run in an Illumina HiSeq2000 platform using a 100 bp paired-end setup. For MethylCap-seq, TruSeq genomic DNA libraries were prepared and samples were run in an Illumina HiSeq platform using a 100 bp paired-end setup. For ChIP-seq, TruSeq genomic DNA libraries were prepared and samples were run in an Illumina HiSeq platform using a 100 bp paired-end setup.