title: GSM1859508: TAB-seq_mm_E9_5; Mus musculus; OTHER
library_construction_protocol: For DNA extraction, embryos were lysed in 20 mM Tris pH 8.0, 100 mM NaCl, 15 mM EDTA, 1% SDS, 0.5 mg/ml proteinase K, for 3h at 55ºC. This was followed by two Phenol/Chlorophorm/Isoamylalcohol (25:24:1, PCI) extractions and subsequent centrifugation (5 minutes at 13,000 rpm). The DNA was then precipitated by adding 1/5 volumes of 4 M NH4Ac and 3 volumes of 96% ethanol. The reaction was left on ice for a minimum of 30 minutes. The DNA precipitate was centrifuged for 20 minutes at 4°C (13,000 rpm) and the pellet was washed with 500 μL of 70% ethanol and centrifuged for 5 minutes (13,000 rpm) at room temperature. The pellet was then resuspended in 200 μL of TE buffer and 1 μl of RNAse A (20 μg/μl) was added. The reaction was left to proceed for 30 minutes at room temperature after which it was precipitated with 0.1 volumes of 4 M NH4Ac and 1 volume of isopropanol on ice for 2 h. The reaction was then centrifuged for 30 minutes at 4°C (13,000 rpm) and the pellet resuspended in TE buffer. TAB-seq library generation was performed with the 5hmC TAB-seq Kit (WiseGene, USA - Cat# K001) as per manufacturer's instructions, with minor modifications related to the amount of starting material (100 ng).  
attributes.tag: GEO Accession
attributes.value: GSM1859508