title: GSM4057527: tet2 wt/wt_meDIP_rep2; Danio rerio; MeDIP-Seq
library_construction_protocol: Kidney marrow extracted from adult zebrafish, followed by single-cell suspension generation by vigrous pipetting and passing through 70 micron filter. Genomic DNA was sonicated to 200bp with a Covaris E220 Focused-ultrasonicator followed by end repair for 30 minutes at room temperature.  A-tailing was performed for 30 minutes at 37°C and adapters were ligated overnight at room temperature.  SPRI bead clean-ups were performed after end repair, a-tailing, and adapter ligation.  Three spike-in DNA controls were used to assess immunoprecipitation (IP) specificity: unmethylated ƛ, fully methylated T7, and fully hydroxymethylated M13.  Spike-in controls were sheared into libraries and adapter ligated as previously described. Spike-in controls were added in a 1:1000 ratio to the sample libraries prior to template denaturation.  100ng (meDIP) and 150ng (hmeDIP) adapter ligated libraries were denatured at 95°C for 10 minutes and snap-cooled on ice for two minutes to allow antigen accessibility.  5ng of each sample library (with spike-in added) was removed and reserved as input (no-IP) control DNA.  Primary IP reactions were performed overnight at 4°C with overhead rotation using anti-5-methylcytosine antibody (Eurogentec BI-MECY-0100) or anti-5-hydroxymethylcytosine antibody (Eurogentec BI-HMCY-0100).  Secondary IP reactions were performed for 2 hours at 4°C with overhead rotation with M-280 sheep anti-mouse Dynabeads (ThermoFisher 11201D).  The library-bound magnetic beads were washed on a magnet six times with IP buffer (10mM Sodium Phosphate buffer, 140mM NaCl, 0.05% Triton X-100) to remove non-specific fragments.  Proteinase K digestion in TE buffer with 0.25% SDS was performed at 55°C for 2 hours with gentle rotation on a Thermomixer (1250 rpm).  Samples were purified with a SPRI bead clean-up and the reserved input (no-IP) control DNA wells were added back to the sample plate before PCR.  To complete library generation, a 7-cycle PCR reaction with Phusion HotStart II DNA Polymerase (ThermoFisher) was performed to add i7 indices and Illumina flow cell adapter sequences.  Qubit High Sensitivity DNA quants, Agilent Bioanalyzer High Sensitivity DNA traces, and qPCR against spike-in controls (unmethylated ƛ, fully methylated T7, and fully hydroxymethylated M13) were used as quality control (QC) metrics to assess the quality of the final libraries.  Libraries that passed QC were pooled and sequenced as paired-end 125nt reads on the Illumina HiSeq 2500 platform.
attributes.tag: GEO Accession
attributes.value: GSM4057527