title: GSM4648849: hMGL SORL1-A528T H3K27ac Rep2; Homo sapiens; ChIP-Seq
library_construction_protocol: hMGLs were detached by accutase digestion and rinsed twice with PBS. Cells were pelleted in 1.5 ml Eppendorf tubes and fixed using 1 ml 1% formaldehyde (Sigma, F8775, St Louis, MO) diluted in PBS at room temperature for 10 min. Formaldehyde was quenched by adding a Glycine solution to a final concentration of 125 mM and incubated for 5 min at room temperature. After washing twice with 1 ml PBS, 1 ml cell lysis buffer was added to the cell pellet, incubated for 10 min on ice, and further lysed in lysis buffer (iL2). Chromatin was resuspended in 130 ml Shearing buffer and sheared using a Covaris S220 sonicator to 200-500 bp (Duty cycle 2% for 10 min) in an M220 microTUBE (Covaris, 520077). Equal aliquots of sonicated chromatin were used per immunoprecipitation reaction comprising 2 ml H3K27ac antibody (Abcam, Ab4729), H3K4me3 antibody (Millipore, 07-472), or 2 ml Rabbit IgG control antibody (Diagenode) and 1 ml sheared chromatin as input. The total volume of each ChIP reaction was 300 ml, and included 6 ml 5% BSA, 1.5 ml 200 x protease inhibitor cocktail, 56 ml 5 x buffer iC1, 20 ml magnetic beads, 2 ml antibody and sonicated chromatin. ChIP reactions were incubated overnight at 4 °C under constant rotation. Magnetic beads (conjugated with antibody-histone-DNA) were washed with 350 ml buffer iW1, iW2, iW3, and iW4. Precipitated chromatin was eluted in 100 ml Elution buffer iE1 for 30 min at room temperature under constant agitation, and transferred into a 200 ml tube. Chromatin crosslinks were uncoupled by adding 4 ml of Elution buffer iE2 and incubating for 4 h at 65°C. DNA was isolated using IPure beads v2 and eluted in 25 ml Elution buffer. 10 ml precipitated DNA was further processed for template preparation, library synthesis, and library amplification. Amplified products were purified, and size selected using AMPure XP beads (Beckman Coulter, A63880). All libraries were quantified using an Agilent 2100 Bioanalyzer Samples were pooled/sequenced using a NextSeq 500/550 High Output v2 kit using paired-end reads on a NextSeq 500 platform (Illumina).
attributes.tag: GEO Accession
attributes.value: GSM4648849