Sample information curated by ChIP-Atlas

Antigen

Antigen Class
Input control
Antigen
Input control

Cell type

Cell type Class
Pluripotent stem cell
Cell type
hESC H9
NA
NA

Attributes by original data submitter

Sample

source_name
H9 Human ESCs-derived PAX6 positive cells
cell type
H9 Human ESCs-derived PAX6 positive cells
passages
p21-50
chip antibody
none

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Immunoprecipitated DNA or Input DNA for each cell cycle phase, cellular condition (ESC vs PAX6 cells), and biological replicate were end repaired using the End-It DNA End-Repair Kit (Epicenter), extended using a Klenow fragment (3′-5′ exo) (Epicenter), and ligated to sequencing adaptor oligos (Illumina) by following manufacturers´ recommendations. Each library was then subjected to 15 cycles of PCR amplification using PFU Ultra II Hotstart Master Mix (Agilent), and a size selected in range of 300 ± 50 bp. The final libraries were quantified by using both Qubit fluorimeter (Life Technologies) and Bioanalyzer system (Agilent Technologies), and submitted for sequencing. Before every cell sorting experiment, cells were allowed to establish robust colonies. Normally, they were collected 4-5 days after plating. To improve the yield of sorting, we synchronized hESCs at either G2/M or G1 phase of the cell cycle one day before the collection, by incubation with Nocodazole (Sigma–Aldrich, Catalog number M1404-2MG) (200 ng/ml for 16 hr when cells were grown in mTeSR-1) (Figure 2B). Blocking of hESCs in G2/M phase was reversible, and cells were able to resume the cell cycle and progress into G1 after withdrawing of Nocodazole from the culture medium (Figure 1C). For cells growing in E8 medium, 25 ng/ml of Nocodazole for 16 hr was enough to block most of the cells in G2/M and allowed progression into G1 after withdrawing of Nocodazole from the medium (data not shown). After synchronization, cells were collected as single cells by treatment with Acutase (MP Biomedicals, Catalog number 1000449), and subsequently fixed with 1% formaldehyde for 10 minutes followed by 5 minutes of incubation with 0.125M glycine (Sigma-aldrich, Catalog number G8790-100G). Cells were counted and stored at -80ºC. Pure populations of cells at G2, mitosis or G1 phase of the cell cycle were isolated by FACS, taking advantage of differences in DNA content that allow us to distinguish cells in G2/M from cells in G1, and the exclusive presence of histone H3 phosphorylated in serine 28 (H3S28p) in mitosis that permits us to discriminate between cells in G2 or M phase. Subsequent to cell synchronization and fixation, cells were permeabilized for 10 minutes using a mild permeabilization/wash buffer containing Saponin (BD Bioscience, Catalog number 51-2091KZ). For ESCs isolation, staining was performed with antibodies against OCT4 (PE-conjugated, BD Bioscience, Material number 561556), H3S28p (Alexa fluor 647-conjugated, BD Bioscience, Material number 558609), for 30 min. For PAX6 cells, staining was performed with antibodies against PAX6, instead of OCT4 (PE-conjugated, BD Bioscience, Material number 561552). After staining, cells were washed with permeabilization/wash buffer, resuspended in 2% fetal bovine serum (FBS) in phosphate buffer saline (PBS) and counterstained with 1 μg/ml DAPI (Life Technologies, Catalog number D1306) for 30 min. FACS was performed on a BD FACSAria II using linear FSC and SSC scaling, followed by height and area-based doublets discrimination. Compensation was calculated using FACS Diva autocompensation algorithms, and supplemented by manual compensation to correct for autofluorescence. Cells with sub-normal DNA content were filtered out during the gating and only samples with a purity of 95% or higher were used in the following experiments. Cells at specific phases of the cell cycle, obtained from 3-4 different cell-sorting experiments, were pooled and treated as a single sample (or biological replicate). Two biological replicates were analyzed for each cell cycle phase, and for each cellular condition (ESCs and PAX6 cells). Cells were washed twice with PBS and subjected to extraction of nuclei according to (Siegel et al., 2009) with modifications. Isolated nuclei were sonicated using a Misonix S-4000 Ultrasonic Processor (QSonica,) to obtain sheared chromatin ranging from 0.2 to 0.6 kilobases (Figure S1). 4 μg of sheared chromatin were immunoprecipitated with either anti H3K4me3 (Abcam, ab1012) or anti H3K27me3 (Millipore, 07-449). Immunoprecipitated complexes were purified by Protein-G Dynabeads (Invitrogen), eluted, reverse crosslinked, quantified, and subjected to library preparation.

Sequencing Platform

instrument_model
Illumina Genome Analyzer II

hg38

Number of total reads
62761054
Reads aligned (%)
96.0
Duplicates removed (%)
2.2
Number of peaks
1832 (qval < 1E-05)

hg19

Number of total reads
62761054
Reads aligned (%)
95.2
Duplicates removed (%)
3.9
Number of peaks
1834 (qval < 1E-05)

Base call quality data from DBCLS SRA