GSM4983816: Input (IN); Escherichia coli KRX; ChIP-Seq
The lysates were passed three times in a French press at 12,000 psi and each lysate was heated for 10 minutes at 50°C to denature free Tus-GFP. Supernatants were immunoprecipitated (excluding input) with goat anti-GFP IgG (Abcam; Ab6673) and Immunocaptured DNA was released by adding elution and de-crosslinking buffer containing proteinase k. Samples were incubated at 95°C to denature proteinase K and residual crosslinked proteins. Each library was prepared using the NEBNext Ultra DNA library preparation kit for Illumina.Briefly, 55.5 µL of DNA suspensions were end repaired. Due to the low DNA concentrations in the suspensions, the NEB adaptors were diluted 10 fold in water to 1.5 µM for ligation as recommended. The adaptors were cleaved using uracil excision. Size selection was not recommended for sample < 50 ng. DNA was then cleaned up using Sera-Mag beads (ratio of 1.4) and eluted in 28 µL of 0.1xTE. Index primers were added by PCR using 18 cycles.  The samples were pooled in a single library, denatured and loaded for sequencing with an Illumina MiSeq desktop sequencer.