Sample information curated by ChIP-Atlas

Antigen

Antigen Class
TFs and others
Antigen
MECP2

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
WT
chip antibody
anti- MeCP2

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)

Sequencing Platform

instrument_model
Illumina HiSeq 2500

hg19

Number of total reads
79496388
Reads aligned (%)
167.2
Duplicates removed (%)
8.0
Number of peaks
818 (qval < 1E-05)

hg38

Number of total reads
79496388
Reads aligned (%)
169.9
Duplicates removed (%)
7.7
Number of peaks
2074 (qval < 1E-05)

Base call quality data from DBCLS SRA