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| Status |
Public on May 01, 2013 |
| Title |
Hepatoblast derived from HUES64 RNA-Seq reps1and2 |
| Sample type |
SRA |
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| Source name |
in vitro derived hepatoblast
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| Organism |
Homo sapiens |
| Characteristics |
cell type: HUES64 derived hepatoblast
chip antibody: NA
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| Growth protocol |
Human embryonic stem cells were expanded on murine embryonic fibroblasts (Global Stem) in KO-DMEM (Life Technologies) containing 20% Knockout serum replace (Life Technologies) and FGF2 (10 ng/mL) (Millipore). Cultures were passaged by enzymatic dissociation using Collagenase IV (1mg/mL) (Life Technologies). Prior to differentiation, cells were plated on matrigel-coated plates (BD Biosciences) and cultured in mTeSR1 (Stem Cell Technologies) for 3 to 4 days. Endoderm differentiation was induced in Advanced RPMI (Invitrogen), 0.5% FBS (Hyclone), Activin A (100ng/mL) (R&D) and WNT3A (50 ng/mL) (R&D). HUES64- derived hepatoblasts (dHep) were induced by culturing day 5 endoderm in RPMI media containing B27(1X), FGF2 (10ng/mL)(Millipore) and BMP4(20ng/mL)(R&D) for five days, and collected after 10 days total of differentiation.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Cells collected by FACS were crosslinked in 1% formaldehyde for 15 minutes at room temperature, with constant agitation, followed by quenching with 125mM Glycine for 5 minutes at room temperature with constant agitation. Nuclei were isolated and chromatin was sheared using Branson sonifier until the majority of DNA was in the range of 200-700 base pairs. Chromatin was incubated with antibody overnight at 4°C, with constant agitation. Co-immunoprecipitation of antibody-protein complexes was completed using Protein A or Protein G Dynabeads for 1 hour 4°C, with constant agitation. ChIPs were completed using previously reported methods (Mikkelsen et al., 2010)
ChIP-Bisulfite Sequencing (adapted from Brinkman et al, 2012) DNA was first subjected to end-repair in a 30-μl reaction containing 6 units T4 DNA polymerase, 2.5 units DNA Polymerase I (Large Klenow Fragment), 20 units T4 Polynucleotide Kinase (all New England Biolabs), dATP, dCTP, dGTP, and dTTP (0.125 mM each), and 1× T4 Ligase buffer with ATP for 30 min at 20°C. DNA was then adenylated in a 20-μl reaction containing 10 units Klenow Fragment (3′→5′ exo-) (New England Biolabs), 0.5 mM dATP and 1× NEB buffer 2 for 30 min at 37°C. DNA was then ligated to preannealed Illumina genomic DNA adapters containing 5-methylcytosine instead of cytosine (ATDBio) using T4 DNA ligase (New England Biolabs). Adapter-ligated DNA fragments were subsequently purified by phenol extraction and ethanol precipitation and size-selected on gel. 50 ng sheared and dephosphorylated Escherichia coli K12 genomic DNA was added to adapter-ligated DNA as carrier during size-selection and bisulfite conversion. DNA was run on 2.5% Nusieve 3:1 Agarose (Lonza) gels. Lanes containing marker (50 bp ladder; New England Biolabs) were stained with SYBR Green (Invitrogen), and size regions to be excised were marked with toothpicks and adapter-ligated DNA fragments from 200–400 and 400–550 bp were excised. DNA was isolated from gel using the MinElute Gel Extraction kit (QIAGEN). The low and high libraries were kept separate in subsequent steps. Adapter-ligated and size-selected DNA was subjected to two subsequent 5-h bisulfite treatments using the EpiTect Bisulfite kit (QIAGEN) following the manufacturer's protocol for DNA isolated from FFPE tissue samples. PCR amplification was done with 1.25 units Pfu Turbo Cx Hotstart DNA Polymerase (Stratagene), primer LPX 1.1 and 2.1 (0.3 μM each), dNTPs (0.25 mM each), 1× Turbo Cx buffer. Amplified libraries were purified with the MinElute PCR Purification kit (QIAGEN) and subsequently purified from gel essentially as described above; whole gels were stained with SYBR Green, and no carrier DNA was added. Final libraries were analyzed on analytical 4%–20% TBE Criterion precast gels (BioRad), and measured by Quant-iT dsDNA HS Assays (Invitrogen). CHIP:Cells collected by FACS were crosslinked in 1% formaldehyde for 15 minutes at room temperature, with constant agitation, followed by quenching with 125mM Glycine for 5 minutes at room temperature with constant agitation. Nuclei were isolated and chromatin was sheared using Branson sonifier until the majority of DNA was in the range of 200-700 base pairs. Chromatin was incubated with antibody overnight at 4°C, with constant agitation. Co-immunoprecipitation of antibody-protein complexes was completed using Protein A or Protein G Dynabeads for 1 hour 4°C, with constant agitation. ChIPs were completed using previously reported methods (Mikkelsen et al., 2010). Sequencing library production details can be found in the Supplemental Experimental Procedures. Sequencing libraries were submitted for sequencing on the Illumina Hiseq 2000. Immunoprecipitated DNA was end repaired using the End-It DNA End-Repair Kit (Epicentre), extended using a Klenow fragment (3’-5’ exo)(NEB), and ligated to sequencing adapter oligos (Illumina). Each library was then PCR-amplified using PFU Ultra II Hotstart Master Mix (Agilent), and a size range of 300-600 was selected for sequencing. RNA-Seq: Strand specific libraries were constructed as described in the main text using a strand specific method (Levin et al., 2010). Polyadenylated RNA was isolated using Oligo dT beads (Invitrogen) and fragmented to 200-600 base pairs, and then ligated to RNA adaptors using T4 RNA Ligase, (NEB), preserving strand of origin information. A primer that anneals to the RNA adaptor was used to facilitate cDNA synthesis, which was then followed by RNA degradation. The cDNA library was then ligated to a DNA adaptor, which was used for PCR enrichment of the library, with index sequences included in the primers used for amplification.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
BiSeq: raw sequencing reads were aligned using maq in bisulfite mode (Li et al. 2008) against human genome version hg19/GRCh37, discarding duplicate reads. DNA methylation calling was performed based on an extended custom software pipeline published previously for RRBS (Gu et al., 2010).
RNA-Seq: raw sequence reads were aligned using Tophat v2.0.6 and Bowtie version 0.12.7.
RNA-Seq: Reads were mapped to the human genome (hg19) using TopHat v2.0.6 (Trapnell et al., 2009)(http://tophat.cbcb.umd.edu) with the following options: “—library-type firststrand” and “—transcriptome-index” with a TopHat transcript index built from RefSeq. Transcript expression was estimated with an improved version of Cuffdiff 2 (Trapnell et al., 2013) (http://cufflinks.cbcb.umd.edu). Cuffdiff was run with the following options: “—min-reps-for-js-test 2 –dispersion-method per-condition” against the UCSC iGenomes GTF file from Illumina (available at http://cufflinks.cbcb.umd.edu/igenomes.html). The workflow used to analyze the data is described in detail in (Trapnell et al., 2012) (alternate protocol B).
ChIP-Seq data was aligned to the hg19/GRCh37 reference genome using bwa version 0.5.7 (Li et al., 2009) with default parameter settings. Subsequently, an improved version of Cuffdiff 2 was used to conduct de novo transcript assembly and RNA expression quantification. Following assembly, transcripts were annotated using known RefSeq genes
For OCT4, SOX2, NANOG and FOXA2 aligned read files were processed with macs version 1.4 (Zhang et al., 2008) using the following parameters: -g 2.7e9 --tsize=36 --pvalue=1e-5 --keep-dup=1. HUES64 WCE (GSM772807) was used as input control.
Genome_build: hg19
Supplementary_files_format_and_content: Hepatoblast_genes_fpkm_tracking.gtf file contains transcript coordinates, FPKM counts and detection p-values.
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| Submission date |
Apr 17, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Michael Johannes Ziller |
| Organization name |
Max Planck Institute of Psychiatry
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| Department |
Translational Psychiatry
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| Lab |
Ziller lab
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| Street address |
Kraepelinstrasse 2-10
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| City |
Munich |
| ZIP/Postal code |
80804 |
| Country |
Germany |
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| Platform ID |
GPL16791 |
| Series (1) |
| GSE46130 |
Transcriptional and Epigenetic Dynamics During Specification of Human Embryonic Stem Cells |
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| Relations |
| BioSample |
SAMN02046822 |
| SRA |
SRX266864 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1124072_Hepatoblast_genes_fpkm_tracking.gtf.gz |
751.8 Kb |
(ftp)(http) |
GTF |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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