GSM8727549: Donor_2_d6_LPS_H3K18lac; Homo sapiens; ChIP-Seq
Purified cells were fixed with 1% methanol-free formaldehyde and quenched with glycine. Fixed cell preparations were lysed, and equal amounts of chromatin were incubated with 1µg of H3K18la antibody (PTM-1406; PTM Biolabs), and incubated overnight at 4ºC with rotation. Dynabeads protein A/G magnetic beads , were washed, centrifuged, mixed with the samples (20 µL/sample) and rotated for 1 hour at 4°C. The bead-bound chromatin complexes  were washed with 400µl buffer for 10 minutes at 4°C with five rounds of washes. After washing chromatin was eluted in 200µl of 0.1 M NaHCO3, 1% SDS elution buffer for 20 minutes in RT. Supernatant was collected, and incubated with proteinase K  for 4 hours at 65°C to disrupt cross‐links between proteins and DNA. Finally samples were purified using Qiaquick MinElute PCR purification Kit (Qiagen)  and eluted in 20µl EB. ChIP-seq libraries were prepared using the Kapa Hyper Prep Kit according to manufacturer's protocol, with the following modifications. 2.5 mL of the NEXTflex adaptor stock (600 nM, Bio Scientific) was used for adaptor ligation of each sample. Libraries were amplified with 12-15 PCR cycles followed by a double post-amplification clean-up was used to ensure proper removal of adapters. Libraries were paired-end sequenced to a read length of 50 bp on an Illumina NextSeq500.