title: GSM2551848: HFD1; Mus musculus; Bisulfite-Seq
library_construction_protocol: Denuded zygotes (30 zygotes from 5 mice for each sample) were collected following removal of the zona pellucida with acidic Tyrode’s solution and removal of polar bodies following a Piezo pulse. Samples were lysed in lysis buffer (10 mM Tris-Cl pH 7.4 and 2% SDS) with 0.5 μl protease K for 1 h at 37 °C. Bisulfite conversion was performed on cell lysates using EZ DNA Methylation-Gold Kit (Zymo). Purification was performed and DNA was eluted in 10 mM Tris-Cl and combined with oligo 1. Klenow were added and incubated at 4 °C for 5 min, 37 °C for 30 min. Following incubation at 95 °C for 1 min, samples were transferred to ice before addition of fresh oligo 1. This random priming and extension was repeated a further three times. Samples were then incubated with 40 U exonuclease I and M-280 Streptavidin Dynabeads. After washes and resuspension in oligo 2 and Blue Buffer, samples were incubated at 95 °C for 45 s, transferred immediately to ice before addition of 100 U Klenow. Libraries were amplified by PCR, and assessed for quality and quantity using Agilent Bioanalyzer and StepOnePlus Real-Time PCR System. Libraries were prepared for 125-bp paired-end sequencing on a HiSeq2500.
attributes.tag: GEO Accession
attributes.value: GSM2551848