title: GSM2310113: H2Y35ADXY_2_160202_BDI--002_Mouse_ATGTCA_R_160201_WENQU_M002; Mus musculus; Bisulfite-Seq
library_construction_protocol: DNA samples were extracted using the QIAamp DNA KIT, from QUIAGEN. For bisulphite conversion, the details are explained below. DNA samples were prepared for whole genome bisulphite sequencing using the TrueMethyl® Whole Genome Kit (CEGX®) according to the manufacturer’s instructions. Briefly, 200 ng of genomic DNA was sheared to 800 bp using M220 Focused-ultrasonicator™(Covaris®). Then the fragmented DNA was denatured and oxidised by a chemical oxidant to convert 5-hydroxymethyl cytosine to 5-formylcytosine (5fC). The purpose of the oxidation was to ensure that we purely captured the information for 5’ methyl cytosine methylation and not an indistinguishable pattern combination of 5’methyl cytosine and 5’hydroxymethyl cytosine. Following oxidation, the DNA was subjected to bisulphite conversion for the deamination of cytosines and 5fC to uracils. Bisulphite converted DNA was desulfonated and purified to then proceed to library preparation. In this “post-bisulfite conversion” library preparation method, the fragmented single stranded bisulfite converted DNA was adapted with sequencing adaptors at the 3’end followed by an extension step and finally ligation of adaptors at the 5’ end of the molecules. Finally, the libraries were indexed and amplified. The PCR was performed for 10 cycles followed by bead-based purification. An additional purification and size selection step using Agencourt AMPure XP beads (Beckman Coulter, Cat: A63881) was performed to remove adaptor dimers. The purified library was eluted in a final volume of 14 ml pure water. Quality of the library obtained was checked using the DNA High sensitivity chip on the Agilent Bioanalyzer. The library was quantified using Qubit and KAPA Biosystems Library quantification kit (#KK4824) according to manufacturer’s instructions. A total of 12pMol of multiplexed libraries were sequenced on a 125 bp paired-end (PE) format (Illumina HiSeq2500).
attributes.tag: GEO Accession
attributes.value: GSM2310113