GSM4746075: 22RV1_FOXA1_DMSO_14MIN_2; Homo sapiens; ChIP-Seq
15x107 22RV1 carcinoma cells were treated for 24h with DMSO and were crosslinked by adding formaldehyde to a final concentration of 0.8% to the cells in media. The fixation was terminated by adding Glycine to a final concentration of 125mM Glycine on a rocker at RT. The cells were washed in ice cold PBS buffer thrice and the cells were harvested using a cell lifter. The crosslinked cells were then lysed in L1 buffer (50mM Hepes, 140mM NaCl, 1mM EDTA, 10% Glycerol, 0.5% Igepal and 0.25% Triton X 100) with protease inhibitor (PI) at 4C in a rotor. The cells were pelleted and resuspended in L2 buffer (200mM NaCl, 1mM EDTA, 0.5mM EGTA in 10mM Tris) with PI and incubated at RT in a rotor. The nuclei were pelleted by centrifugation at 4°C and were washed twice with the sonication buffer (SE) (10mM Tris, 1mM EDTA, 0.1% SDS). The pellet was resuspended in SE with PI and was sheared using a covaris LE220 sonicator (Covaris, Woburn, MA). Library preparations for the ChIP samples were performed as described in Buenrostro et al., 2013 with minor changes. In brief to generate multiplexed libraries, A preamplification cycle was performed with the transposed DNA. The DNA was amplified for 5 PCR cycles with Illumina indexed primers (Nextera index kit, Illumina), 25ul of NEBNext High-Fidelity 2x PCR Master Mix (New England Biolabs). 5ul of this amplified DNA was amplified further using a qPCR to determine the appropriate number of cycles of PCR cycles needed to achieve maximum amplification with minimum duplicates. The number of cycles needed was calculated by finding the cycle number that corresponds to 1/4th of the maximum fluorescence intensity. The final amplification was performed in the leftover 45ul from the preamplification reaction using the number of cycles calculated in the qPCR.