title: GSM8297058: yqjI native 2; Escherichia coli K-12; ChIP-Seq
library_construction_protocol: TF-DNA complexes were fixed with formaldehyde and glycine.  For in vivo ChIP-Seq, cells were sheared via sonication; for in vitro ChIP-Seq, DNA was sheared via sonication prior to TF-DNA binding.  Sheared DNA was immunoprecipitated (IP) using anti-FLAG, anti-V5, or anti-6xHis mAb (indicated in metadata).  DNA was washed with IPP150 buffer and purified via ProteinaseK digestion and Qiaquick PCR purification kit (Qiagen - Cat. No. 28106).  For Library-ChIP, preIP samples used sheared DNA purified via ProteinaseK digestion and Qiaquick PCR purification kit prior to addition of anti-tag antibody; postIP samples used sheared DNA that was immunoprecipitated, washed, purified, and extracted in the same way as ChIP-Seq. ChIP-DNA was converted into NGS libraries compatible with the Illumina platform using the NEBNext Ultra DNA (NEB Cat. No. E7465).  For Library-ChIP, libraries were constructed similarly to Illumina's 16S metagenomic library prep (Illumina Ref #15044223 Rev. B).  Briefly, an initial PCR was performed on DNA with a universal primer pair targeting flanks of the insertion site on the pAMD plasmid where designed sequences were inserted.  These primers contained overhangs that could be targeted by Illumina Nextera XT Index Kit v2 (Illumina Cat. No. FC-131-2001) primers.  First PCR was cleaned up with Qiaquick PCR purification kit (Qiagen - Cat. No. 28106).  Second PCR used a unique pair of primers from the Illumina Nextera kit for each sample, and were cleaned up with AMPureXP beads (Fisher Scientific Cat. No. NC9933872).