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| Status |
Public on Dec 23, 2015 |
| Title |
PAX6-R2_RNA |
| Sample type |
SRA |
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| Source name |
H9 Human ESCs-derived PAX6 positive cells
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| Organism |
Homo sapiens |
| Characteristics |
cell type: H9 Human ESCs
passages: p21-50
chip antibody: none
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| Growth protocol |
The H9 human ESC line from WiCell Research Institute (Madison, WI) was maintained on hESC-qualified Matrigel (BD Bioscience, Catalog number 354277) in mTeSR-1 medium (Stemcell Technologies, Catalog number 05850) or Essential E8 medium (Life Technologies, Catalog number A1517001), as recommended by the supplier. Cells were expanded every 5–6 days, using non-enzymatic passaging following WiCell standard protocols. To generate PAX6 cells, undifferentiated ESCs were incubated in mTeSR-1 medium supplemented with 10 mM of Retinoic Acid (Sigma-Aldrich, Catalog number R2625-50MG) for 5 days. The treatment started one day after plating the cells, and medium was changed every day. hESC research was approved by the Institutional Embryonic Stem Cell Research Oversight Committee at University of Vermont.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNAs from undifferentiated H9 cells or PAX6 cells, were isolated by using Trizol (Invitrogen). Subsequently, we removed DNA contaminants by using DNA-free RNA extraction kit (Zymo Research). Following Illumina’s manual, 2 µg of DNA-free total RNAs was used to construct each paired-end RNA-Seq library with TruSeq RNA Sample Preparation Kit v2 (Illumina). Libraries were subjected to 15 cycles of amplification with pair-end PCR primers (Illumina). Libraries were quantified in Bioanalyzer and sequenced were on the HiSeq-1000 platform (Illumina) for the read length of 100 bases. To address the influence of culture medium on RNA expression, RNA-seq libraries were built from stocks of ESCs maintained in either mTeSR-1 or Essential E8 medium. The RNA-seq libraries for PAX6 cells were generated from original cellular stocks maintained in either mTeSR-1 or E8. No major differences in RNA expression were detected among different stocks. Therefore, libraries of similar cellular condition (Control or Differentiated) were considered biological replicates. R1: mTeSR medium; R2: E8 medium.
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.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 1000 |
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| Data processing |
ChIP-Seq
Base calls and sequence reads were generated by Illumina CASAVA software (version 1.6, Illumina).
Reads were mapped to the human genome (GRCh37, hg19) using bowtie (version 0.12.8) with up to two mismatches allowed.
For each cellular condition (i.e. defined by cell cycle phase and differentiation state), genome-wide enrichment profiles were generated from pooled ChIP-seq replicates versus input for H3K27me3 and H3K4me3, respectively using SPP. The resulting enrichment profiles are conservative (alpha=0.01) statistical estimates of log2 fold-enrichment across the genome.
Genome_build: hg19
Supplementary_files_format_and_content: ChIP-Seq: BigWig files correspond to enrichment profiles that are conservative (alpha=0.01) statistical estimates of log2 fold-enrichment across the genome.
RNA-Seq
Base calls and sequence reads were generated by Illumina CASAVA software (CASAVA 1.8 pipeline, Illumina).
Raw sequences from RNA-seq libraries were mapped using TopHat (version 2.0.8b) with the parameters of “--b2-sensitive -r 80 -g 10”.
Mapped reads were analyzed using SeqMonk software (v0.27.0; Babraham Bioinformatics Institute). Expression profiles were calculated using the RNA-seq quantitation pipeline and normalized by adjusting globally count distributions at the 75th percentile. Differential expression was calculated using the intensity difference statistical test included in the software. Differential expression was called on log2 transformed counts by selecting transcripts that changed with a significance of p < 0.05 after Benjamini and Hochberg correction using a null model constructed from the 1% of transcripts showing the closest average level of observation to estimate experimental noise.
Genome_build: hg19
Supplementary_files_format_and_content: RNA-Seq: Tab-delimited text files include RPKM values for each sample.
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| Submission date |
Mar 03, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Troy W. Whitfield |
| Organization name |
University of Massachusetts Medical School
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| Street address |
55 Lake Ave. N.
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| City |
Worcester |
| ZIP/Postal code |
01655 |
| Country |
USA |
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| Platform ID |
GPL15433 |
| Series (1) |
| GSE55502 |
Unique cell cycle-dependent variations in the pluripotent epigenetic landscape define novel cohorts of temporal expressed bivalent genes during hESCs differentiation |
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| Relations |
| BioSample |
SAMN02670989 |
| SRA |
SRX480627 |
| Supplementary data files not provided |
SRA Run Selector |
| Processed data are available on Series record |
| Raw data are available in SRA |
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