Sample information curated by ChIP-Atlas

Antigen

Antigen Class
TFs and others
Antigen
MTF2

Cell type

Cell type Class
Pluripotent stem cell
Cell type
NT2-D1
Tissue
testis
Lineage
inner cell mass
Description
malignant pluripotent embryonal carcinoma (NTera-2), "The NTERA-2 cl.D1 cell line is a pluripotent human testicular embryonal carcinoma cell line derived by cloning the NTERA-2 cell line." - ATCC. (PMID: 6694356)

Attributes by original data submitter

Sample

source_name
Embryonic stem cell and NT2 cell line
cell type
Embryonic stem cell and NT2 cell line
genotype
Wild type
antibody
PCL2

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Embryonic stem cells were washed once with PBS before crosslinking for 10 minutes with PBS containing 1% formaldehyde (Sigma). Crosslinking was quenched with 0.125M Glycine for 5 minutes before two PBS washes. The crosslinked cells were lysed in 6mL of SDS-Lysis buffer (100mM NaCl, 50mM Tris pH8.1, 5mM EDTA pH 8.0, 0.02% NaN3, 0.5% SDS, 2μg/mL Aprotonin, 1μg/mL Leupeptin, 1mM PMSF). Chromatin was pelleted by centrifugation at 1200rpm for 5 minutes at room temperature. The supernatant was then discarded, and the chromatin was resuspended in 3mL of ChIP buffer (2:1 dilution of SDS-Lysis buffer: Triton dilution buffer [100mM Tris pH 8.6, 100mM NaCl, 5mM EDTA pH 8.0, 0.02% NaN3, 5% Triton X-100, 2μg/mL Aprotonin, 1μg/mL Leupeptin, 1mM PMSF]). Chromatin was sheared to approximately 200bp-600bp fragments by sonication on a Branson Sfx150 Sonifier for a total of 4min at 50% amplitude. Sonicated chromatin was incubated overnight with antibody while rotating at 4°C. Following clarification, the chromatin was incubated for 3 hours with 50µL of protein A or G Dyna beads (ThermoFisher) beads. After incubation, the beads were washed three times in Mixed Micelle Buffer (150mM NaCl, 20mM Tris pH 8.1, 5mM EDTA pH 8.0, 5.2% Sucrose, 0.02% NaN3, 1% Triton X-100, 0.2% SDS), twice with Buffer 500 (0.1% Sodium Deoxycholate, 1mM EDTA pH 8.0, 50mM HEPES pH7.5, 1% Triton X-100, 0.02% NaN3), twice with LiCl detergent wash (0.5% Sodium Deoxycholate, 1mM EDTA pH 8.0, 250mM LiCl, 0.5% NP-40, 10mM Tris pH 8.0, 0.02% NaN3) and finally one wash with TE. Immunoprecipitated material was eluted from the beads with Elution buffer (0.1M NaHCO3, 1% SDS) while shaking for 1 hour at 65°C. The supernatant was retained and incubated overnight at 65°C while shaking to reverse the crosslinks. The eluted complexes were then subject to RNase (Thermo Fisher) and Proteinase K (Sigma) treatment prior to DNA clean up (Qiagen Qiaquick PCR Purification Kit). ChIP enrichments were analysed by qPCR using the SYBR Green I detection chemistry (M3003E NEB) on an Applied Biosystems Quant Studio 3 platform. Following the ChIP experiment, the precipitated DNA was quantified using the Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Q32854). A Total of 2-10 ng of DNA from each ChIP-Rx experiment was used for library preparation using the NEBNext Ultra II DNA Library Kit for Illumina (E7645) and NEBNext Multiplex Oligos for Illumina (Set#1, NEB #7335). Following adaptor ligation, DNA was PCR amplified for 5-9 cycles, depending on amount of input DNA. DNA purification was then performed using NEBNext Sample Purification Beads (E7767S). The quality of DNA libraries was analysed on a High Senstivity D1000 Screen Tape (Agilent). The resulting libraries were then used for cluster generation and sequencing using an Illumina NextSeq 500 (ID: NB501524), with 75bp read length.

Sequencing Platform

instrument_model
NextSeq 500

hg19

Number of total reads
26302437
Reads aligned (%)
10.1
Duplicates removed (%)
4.4
Number of peaks
1959 (qval < 1E-05)

hg38

Number of total reads
26302437
Reads aligned (%)
10.2
Duplicates removed (%)
4.0
Number of peaks
1967 (qval < 1E-05)

Base call quality data from DBCLS SRA