GSM3579722: replicate2 ChIP IP OE4x; Homo sapiens; ChIP-Seq
LUHMES-derived neurons at day 9 of differentiation with four levels of MeCP2: KO, WT, OE 4x and OE 11x (see Table S1) were crosslinked with 1% of Formaldehyde (Sigma) in the medium for 10 min at RT and quenched with 2.5 M Glycine (Sigma) for 2 min at RT. Cells were washed with PBS, scraped from the plate and centrifuged. Crosslinked nuclei were isolated using hypotonic buffer (10 mM Tris-HCl pH7.4, 10 mM NaCl, 3mM MgCl2, 0.1% [v/v] Igepal CA-630) and counted using a haemocytometer. Chromatin from ~4x106 nuclei was sonicated for 20 cycles (30 sec ON and 30 sec OFF) on the high power setting using a Bioruptor (Diagenode). Crosslinked and sonicated chromatin was mixed with 60 ng of sonicated Drosophila chromatin (Active Motif) as a spike-in and the mix was incubated overnight at 4 ºC with antibodies against MeCP2 (D4F3, Cell Signalling) plus spike-in antibody (Active Motif). After overnight incubation, magnetic Protein G coated beads (Thermo Scientific) were added and incubated further 4 hours at 4 ºC. Beads were washed and chromatin was reverse-crosslinked overnight at 65 ºC. DNA was then purified using Agencourt AMPure XP (Beckman Coulter) beads. For ChIP-seq library preparation, IPs for each condition were pooled together to achieve 5 ng total DNA as a starting material. For example, 3-4 IPs were pooled together for the KO sample and 2 IPs were pooled for the WT sample. Libraries were prepared using NEBNext Ultra II DNA library Prep kit (NEB) for both IPs and corresponding inputs. NEBNext Ultra II kit (NEB)
GEO Accession
GSM3579722