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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Jan 19, 2014 |
| Title |
DNase_mES_Day6_Intestinal_Endoderm |
| Sample type |
SRA |
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| Source name |
Murine Intestinal Endoderm Cells
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| Organism |
Mus musculus |
| Characteristics |
cell type: mESC-derived Intestinal Endoderm Cells
strain: 129P2/OlaHsd
datatype: DNase-seq
cell sex: F
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| Treatment protocol |
DNase-Seq was performed using adaptations of previous protocols (Sekiya, et al., Genes Dev. 2009). 1-3 biological replicate DNase-Seq experiments were performed at six stages of mouse ES cell differentiation: mouse ES cells, mesendoderm, endoderm, lateral plate mesoderm, pre-pancreatic endoderm, and intestinal endoderm. A detailed protocol is as follows:
Mouse ES cells or differentiated cells of various stages (30-90 million cells) were harvested from 15 cm tissue culture plates, pelleted at 800g for 5 minutes at 4°C and the supernatant aspirated. Cells were washed twice with 2.5 ml/plate of ice-cold PBS and pelleted (800g, 5 minutes, 4°C). The supernatant was aspirated and cells were resuspended 2.5 mL/plate Buffer A containing 15 mM Tris HCl (pH 8), 15 mM NaCl, 60 mM KCl, 1 mM EDTA (Ambion, pH 8), 0.5 mM EGTA (pH 8) and 0.5 mM Spermidine (Sigma-Aldrich). An equal volume of 2X Igepal in Buffer A (earlier samples with a 0.05% Igepal final concentration, later samples with a 0.00165% Igepal final concentration) was added to the resuspended cells and the nuclei were pelleted (800g, 5 minutes, 4°C). The supernatant was aspirated promptly and the cells resuspended in 2.5 mL/plate Buffer A. An aliquot of the resuspended nuclei was taken and counted on a hematocytometer with 1:10 Trypan Blue. Nuclei were pelleted (800g, 5 minutes, 4°C) and either digested immediately as below, or stored in 20 mM Tris HCl (pH 8), 75 mM NaCl, 0.5 mM EDTA (Ambion) 50% glycerol, 0.85 mM DTT, and 0.125 mM PMSF, flash frozen in liquid nitrogen and stored at -80°C for a later digestion.
If frozen, nuclei were washed twice with 2.5 mL/plate Buffer A, pelleted (800g, 5 minutes, 4°C) and the supernatant discarded before proceeding to digestion. Digestion volumes are given for 107 nuclei. Volumes should be scaled as per cell number. Digestion buffer (850 μl per 107 nuclei) was prepared by diluting 10X stock of CaCl2 and NaCl in Buffer A (above) for a final concentration of 6 mM CaCl2 and 75 mM NaCl. Stop buffer (950 μl per 107 nuclei) was prepared with final concentrations as follows: 50 mM Tris HCl (pH 8), 100 mM Na Cl, 0.1 % SDS, 100 mM EDTA (Ambion, pH 8), 10 μg/ml RNase A (Invitrogen), 1 mM Spermidine (Sigma-Aldrich) and 0.5 M Spermine (Sigma-Aldrich). Both digestion and stop buffers were warmed at 37°C. DNase I enzyme was combined with warmed digestion buffer for a final volume of 100 μl per 107 nuclei. If multiple concentrations of DNase I were tested, nuclei were separated equally amongst each concentration. Prior to digestion, a small aliquot was reserved as an undigested control for quantitative PCR analysis. The DNaseI/ digestion buffer mix was warmed in a 37°C water bath for 2 minutes, and the nuclei were gently resuspended in digestion buffer. The warmed DNaseI/ digestion buffer mix was promptly added to the nuclei and digested for 2 minutes at 37°C. Pre-warmed stop buffer was added and the tube inverted multiple times to stop the digestion. Digested samples were transferred to a 55°C incubator for 15 minutes. Proteinase K (Ambion, 20 mg/mL) was added to each sample for a final concentration of 2μg/mL and samples were incubated overnight at 55°C.
DNA fragments were purified by adding an equal volume of phenol:chloroform:isoamyl alcohol to each sample and mixing. The suspension was transferred to a phase-lock tube and centrifuged for 5 minutes at 12-16,000g. The aqueous phase was isolated and combined with 0.1 volumes 3M sodium acetate, 2 volumes absolute ethanol and 1:600 Glycoblue. This mixture was incubated at -20°C for 5-6 hours or at -80°C for 30-45 minutes. DNA was pelleted by centrifugation at 12-16000g for 10-20 minutes and the supernatant aspirated. The DNA pellet was washed with 70% ethanol and re-pelleted by centrifugation at 4000 rpm for 5 minutes. The supernatant was carefully aspirated and the DNA pellet air-dried for 5-10 minutes before resuspending in TE buffer.
To enrich for hypersensitive regions, size selection was performed on the purified fragments by either 2% agarose gel band purification or the Invitrogen E-Gel Agarose System. Fragments were collected in the 175-400 bp region so as to avoid empirically-determined complications by the ~150 bp nucleosomal signal. Size selection enrichment was tested by quantitative PCR using primers (three positive control primers and three negative control primers) for constitutively DNase hypersensitive regions and insensitive regions as positive and negative controls respectively, on both the samples as well as the reserved undigested controls. Size selection enrichment was calculated as follows: Enrichment = 2^((Ct(negative control primer)-Ct(positive control primers))digested, size selected fragments-(Ct(negative control primer)-Ct(positive control primers))undigested control).
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| Growth protocol |
Mouse embryonic stem cell culture and endoderm differentiation was modified slightly from previously published protocols (Sherwood, et al., Mech. Dev. 2011). Undifferentiated 129P2/OlaHsd mESCs were maintained on gelatin-coated plates with mouse embryonic fibroblast (MEF) feeders in mESC medium composed of Knockout DMEM (Life Technologies) supplemented with 15% defined FBS (HyClone), 0.1 mM nonessential amino acids (Life Technologies), 1% Glutamax (Life Technologies), 0.55 mM 2-mercaptoethanol (Sigma) and 1x ESGRO LIF (Millipore). Before differentiation, ESCs were passaged onto gelatin-coated plates for 25 min to deplete MEFs. MEF-depleted ESCs were then seeded at 1x104 cells/cm2 onto gelatin-coated dishes in mESC medium. After 12–24 h, medium was changed to Advanced DMEM (Life Technologies) supplemented with N-2 (Life Technologies), B27 without vitamin A (Life Technologies) and 1% Glutamax. After 44–48 h, medium was changed to Advanced DMEM with 2% FBS, 1% Glutamax, 5 nM GSK-3 inhibitor XV and 50 ng/ml Escherichia coli–derived Activin A (Peprotech) for 24 h to produce mesendoderm. For endoderm differentiation, cells were then fed with Advanced DMEM with 2% FBS, 1% Glutamax, 50 ng/ml Activin A and 1 μM dorsomorphin (Sigma) for 48 h. For intestinal endoderm differentiation, cells at the endoderm stage were fed for 24 h with Advanced DMEM with B-27 supplement without vitamin A, 1% Glutamax and 100 nM GSK-3 inhibitor XV. For differentiation of prepancreatic endoderm, cells at the endoderm stage were fed for 24 h with Advanced DMEM with B-27 supplement without vitamin A, 1% Glutamax, 500 nM retinoic acid (Calbiochem), 50 nM A-83-01 (Calbiochem) and 8 ng/ml Bmp4 (Stemgent). For mesodermal differentiation, cells at the mesendoderm stage were treated for 48 h with 10 ng/ml Bmp4.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Samples prepared into Illumina libraries using the Beckman Coulter Genomics SPRI-works system using custom adapters. 6nt 3’ barcodes were added during PCR enrichment and the resulting fragments were evaluated using Agilent BioAnalyzer 2100. Samples were sequenced one-per-lane using an Illumina HiSeq 2000.
Libraries were diluted to 10nM and then run on qPCR with p5 and p7 primers to determine final flow cell loading concentration, normalized to a PhiX control library. PhiX spike: Each lane was spiked with approximately 0.25-0.5% PhiX control library. Samples were run one-per-lane using Standard Illumina protocols using the following: TruSeq SR/PE Cluster kit V3 cBot, TruSeq SBS Reagent Kit V3, RTA version 1.12.4.2, and Control software version 1.4.8.
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| Library strategy |
DNase-Hypersensitivity |
| Library source |
genomic |
| Library selection |
DNAse |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
Illumina Casava 1.8 processing was used and reads were saved into FASTQ files using the standard 33 offset in ASCII Qscore encoding.
Sequenced reads were directly mapped to mm10 genome using BWA version 0.6.2 with default parameters.
Non-unique and low quality reads (MAPQ<20) were removed before normalization and analysis by the PIQ algorithm.
For each sample PIQ input mapped reads from a BAM file, a list of TF sequence PWMs, and produced a text file indicating putative TF binding sites per PWM. Only those PWMs found to exhibit a significant DNase I profile were included (significance threshold: >100 of the 399 bases within the profile passing 5% FDR).
Genome_build: mm10
Supplementary_files_format_and_content: PIQ output: one text file for each sample indicating putative TF binding sites. These files contain the following columns: Chromosome, TF motif center (beginning), TF motif center (end; equal to prior column if motif has odd length), TF identifier, PWM/Sequence match score, direction of PWM match, and PIQ TF binding score
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| Submission date |
Jan 02, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
David K. Gifford |
| Organization name |
Massachusetts Institute of Technology
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| Department |
Electrical Engineering and Computer Science
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| Lab |
Computer Science and Artificial Intelligence Laboratory
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| Street address |
32 Vassar Street
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| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02139 |
| Country |
USA |
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| Platform ID |
GPL13112 |
| Series (1) |
| GSE53776 |
DNase I hypersensitivity and algorithmic prediction of TF binding in early pancreatic mES directed differentiation |
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| Relations |
| BioSample |
SAMN02567740 |
| SRA |
SRX404486 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1300606_DNase_mES_Day6_Intestinal_Endoderm.PIQ_output.txt.gz |
868.7 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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