GSM4594318: POS Acute Tram H3K27ac Replicate 1; Homo sapiens; ChIP-Seq
Cells were fixed with 1% formaldehyde for 10 minutes at room temperature, then quenched for five minutes using 1 mM Glycine. Media was discarded and cells were scraped on ice using a cell lifter. Cells were centrifuged 3K RPM at 4C, then supernatant was aspirated and the pellet was flash frozen in liquid nitrogen. Antibody was conjugated to Protein A Dynabeads (Life Technologies cat. # 1002D) for 4 hours at 4C in PBS + 0.5% BSA. Crosslinked pellets were resuspended in lysis buffer 1 to permeabilize plasma membrane, and centrifuged for 5 minutes at 4C at 200 x g. Nuclei were resuspended in lysis buffer, and centrifuged for 5 minutes at 4C at 200 x g. Chromatin was resuspended in sonication buffer, sonication beads added and chromatin was sonicated at 4C for 15 cycles; 30 seconds on, 30 seconds off. Beads were then washed three times with a low salt sonication buffer, then washed once with a high salt sonication buffer, then washed once with a LiCl wash buffer, then washed once with TE buffer. Fragmented chromatin was eluted from the beads with elution buffer at 65C for 15 minutes, vortexing every two minutes. Eluted chromatin and Dynabeads were placed on the magnet, and eluted chromatin was transferred to a fresh tube. Crosslinking was reversed by incubating the eluted chromatin at 65C overnight. The following day, 0.2 ug/mL RNase A (Sigma cat. # R4642) was added to the samples and incubated for 1 hr at 37C. Protein in the sample was digested using 0.2 ug/mL Proteinase K (Ambion cat. # 25530-015) at 55C. Fragmented DNA was purified using the Qiagen MinElute Kit (Qiagen cat. # 28004). 50 ng of DNA was used for library preparation using the KAPA stranded HyperPrep Kit (Roche cat. # 07962347001) and Illumina TruSeq Indexed Adapters according to the manufacturer's instructions. Dual size selection was performed after 18 cycles of PCR amplification of the cDNA library according to manufacturer recommendations. A multiplexed pool of 12 libraries was sequenced using the 75-cycle NextSeq 500/550 High Output v2 sequencing kit (Illumina cat. # FC-404-2005) on the Illumina NextSeq500 to yield approximately 3.5 x 107 reads per sample.
GEO Accession
GSM4594318