GSM8395441: 2HE-5NMe H3K27me3; Homo sapiens; OTHER
300,000 FXS neurons were harvested, washed and bound to beads. Neurons-beads were divided into three aliquots for determining binding locations of IgG control, H3K4me3, and H3K27me3. Neurons were permeabilized and the appropriate primary antibodies were utilized on each aliquot separately. Protein A-MNase fusion protein (gifted by the Henikoff lab) was then added to bind to primary antibody locations. Targeted digestion was activated with addition of calcium and occurred for 5 minutes. Cut fragments were then released from insoluble nuclear chromatin and extracted using NucleoSpin columns (Takara) following manufacturer's instructions. Size selection was performed using AMPure XP beads (Agencourt) before library preparation with the Kapa Hyper Prep Kit (Roche) following manufacturer's instructions. End repair and A-tailing were performed before adapter ligation with TruSeq (Illumina) indices. The libraries were amplified and then quality control was performed to ensure that primer dimers were negligible.