GSM3931401: 5hmC_r2_MeDIP; Homo sapiens; MeDIP-Seq
Whole cell DNA was extracted from HEK293T cells using the QIAamp DNA mini kit (QIAgen), according to the manufacturers protocol.The MeDIP protocol was based on Weber et al, 2005 (Weber et al., 2005). Contaminating RNA was removed from both mtDNA and whole DNA with 1 µl RNase A (QIAgen) for 30 min at 60oC with shaking in a total volume made up to 200 µl with ddH2O. RNase was subsequently removed by phenol-chloroform extraction and DNA was precipitated with isopropanol. 2 µg of DNA per MeDIP was sheared on a Covaris S220 focused-ultrasonicator using the Illumina 200 protocol (to yield 200 bp fragments) provided by the manufacturer. Shearing size was confirmed using an Agilent 2100 BioAnalyser and the DNA 7500 LabChipTM. Sheared DNA was diluted to 455 µl in TE buffer and denatured at 95oC for 10 min, followed by ice cooling for 10 min. Then, 51 µl of 10x IP buffer (100 mM Na-Phosphate pH 7.0, 1.4 M NaCl, 0.5 % Triton X-100) and 5 µl of either anti-5mC (Abcam, ab10805) or anti-5hmC (Abcam, ab106918) antibodies were added to the DNA and preincubated with overhead shaking for 2 h at 4oC. 20 µl Protein G Dynabeads (Life Technologies) were prewashed 2x with 800 µl 0.1% BSA in PBS for 5 min at RT with shaking. Beads were collected, resuspended in 40 µl of 1x IP buffer (10 mM Na-Phosphate pH 7.0, 0.14 M NaCl, 0.05 % Triton X-100), and then added to the preincubated antibody-DNA mix. Tubes were incubated at 4oC overnight with overhead shaking, and then beads were washed 3x with 800 µl 1x IP buffer. Beads were resuspended in 250 µl of Proteinase K digestion buffer (50 mM Tris pH 8.0, 10 mM EDTA, 0.5 % SDS) with 7 µl of Proteinase K (Fermentas) and incubated at 50oC for 3 h with shaking. DNA was then extracted using phenol-chloroform extraction followed by isopropanol precipitation (Weber et al., 2005). DNA Libraries were constructed using Illumina TruSeq DNA sample prep kit V2 by the Shanghai Institute for Biological Sciences core facility.
GEO Accession
GSM3931401