GSM3579719: replicate2 ChIP input KO; Homo sapiens; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
Input control
Antigen
Input control
Cell type
Cell type Class
Neural
Cell type
LUHMES
Tissue
Mesencephalon
Attributes by original data submitter
Sample
source_name
LUHMES-derived neurons
div
day 9
passage
15-20
cell line
LUHMES
cell type
LUHMES-derived neurons
genotype
MeCP2 KO
chip antibody
none
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
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)