title: GSM1467529: Human Max protein; Homo sapiens; ChIP-Seq
library_construction_protocol: Full-length open reading frames were cloned into the Gateway pDEST17 (N-terminal His-tag) expression vector by recombinational cloning from previously created pENTR clones. All proteins were produced by over-expression in E. coli BL21 (DE3) cells (New England BioLabs), and purified by FPLC (AKTAprime plus) using HisTrap FF affinity columns (GE Healthcare). Anti-His Western blots were performed to assess protein quality and concentration. Hox and HM-Exd purification conditions, expression constructs, and EMSA conditions have been described previously (Joshi et al., 2010; Noro et al., 2006; Ryoo and Mann, 1999). AbdB (residue 224 to the C-terminus) and Pb (residues 126–306) were cloned in pET14b (Novagen) and pQE9 (Qiagen), respectively. For SELEX EMSA lanes, binding reactions were performed with 200 nM double stranded SELEX library, 67 nM Hox, and 33 nM HM-Exd in a final volume of 30 μl. Parallel DNA binding reactions using 32P labeled probes containing known Hox-Exd composite sites were used to track the mobility of Hox+HM-Exd+DNA complexes. Regions corresponding to the cooperative complex were cut out and eluted. The eluted DNA was purified, concentrated, and amplified by PCR. The PCR products were then purified and divided for the next round of SELEX or Illumina sequencing. Subsequent rounds of selection followed the same structure as the first round of SELEX.