GSM4767254: ChIPSeq_PSEN1_A246E_iPSCNeuron_INPUT 2; Homo sapiens; ChIP-Seq
NDC and PSEN1M146L, and PSEN1A246E (biological replicates, n = 1) hiPSC-derived neurons were processed using the Active Motif ChIP-IT High Sensitivity kit according to the manufacturer's recommendations (Active Motif, Cat. 53040). Briefly, cells were washed with cold 1x PBS and collected in a 1.5 mL tube in PBS, followed by formaldehyde fixation for 10 min at room temperature. After addition of stop solution, cells were incubated at room temperature for 5 min and subsequently washed with 1x PBS Wash Buffer and resuspended in Chromatin Prep Buffer. Following incubation on ice for 10 min, cells were lysed by performing 30 plunges in a chilled dounce homogenizer. Samples were then resuspended in Active Motif ChIP Buffer and chromatin sonication performed using a probe tip sonicator on ice for a total of 10 min per sample (30x 20 sec intervals). Optimal sonicated chromatin fragment lengths were confirmed by agarose gel and used for IP reactions. 500 ng of purified, sonicated chromatin was incubated with 5 µL of H3K4Me3 (Active Motif, Cat. 39159) or H3K27Me3 (Active Motif, Cat. 39155) Rabbit polyclonal antibody overnight at 4C. After de-crosslinking, ChIP DNA was purified using the Qiagen MinElute PCR Purification Kit (Qiagen, Cat. 28004). Libraries for H3K4Me3, H3K27Me3, and 1% Input were generated for NDC and psen1M146L conditions at the UC San Diego IGM sequencing core using Illumina TruSeq LT adaptors (Illumina Cat. FC-121-2001). Sequencing performed on Illumina HiSeq4000 generating Single-Read, 75bp reads with an average of 20 million reads per sample.