GSM1647187: K4E-MEF2B-V5-ChIP-rep2; Homo sapiens; ChIP-Seq
Lysates were clarified from sonicated nuclei and MEF2B-DNA complexes were isolated with antibody. Library construction was carried out on the Bravo liquid handling platform using VWorks Automation Control Software (Agilent Automation). Samples were first subjected to end repair using T4 Polynucleotide Kinase (NEB), T4 DNA Polymerase (NEB) and Klenow DNA Polymerase (NEB) at room temperature for half an hour. DNA was purified using PEG-Sera Mag Speedbeads (Fisher), with 13.87% final PEG concentration.  A-tailing was then performed using Klenow exo minus (NEB) at 37oC for 30 minutes. Products were purified as before. Illumina short sequencing adaptors were ligated to A-tailed product using T4 DNA Ligase (NEB) at room temperature overnight. To remove adaptor dimers and library fragments below 200 bp, products were purified twice using PEG-Sera Mag Speedbeads with 8.89% and 10.91% final PEG concentrations. Adaptor ligated libraries were PCR amplified and barcoded using custom indexing primers, Illumina PCR primer 1.0 and 0.5 U of Phusion Hot Start II (Fisher). The initial denaturation step at 98oC for 30 seconds was followed by 13 cycles of 15 seconds at 98oC, 30 seconds at 65oC and 30 seconds at 72oC, and a final step at 72oC for 5 minutes. Amplified libraries were purified using PEG-Sera Mag Speedbeads with 9.19% final PEG concentration. Libraries were quantified using a Qubit HS DNA assay (Invitrogen) and equal-molar amounts were pooled.  Each pool was quantified for sequencing with Kapa SYBR Fast Complete Universal qPCR kit (Kapa Biosystems). ChIP libraries were sequenced on the Illumina HiSeq 2000/2500 platform using v3 chemistry and HiSeq Control Software version 2.0.10. MEF2B-V5 ChIP libraries were sequenced 8 per lane. Input DNA was sequenced 14 per lane.