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| Status |
Public on Mar 27, 2014 |
| Title |
Input sequencing in endoderm cells |
| Sample type |
SRA |
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| Source name |
endoderm
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| Organism |
Mus musculus |
| Characteristics |
cell line: Ainv15 (ATCC SCRC-1029) with Dox-inducible Cdx2-V5 construct
Stage: endoderm
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| Treatment protocol |
During this last 24 hour step, Doxycycline was added to the culture medium at 2ug/ml to induce the iCdx2 construct.
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| Growth protocol |
Prior to differentiation, ES cells were passaged onto gelatin-coated plates for 25 minutes to deplete MEFs. MEF-depleted ES cells were then seeded at 1 *10^4 cells/cm2 onto gelatin-coated dishes in Advanced DMEM (Life Technologies) supplemented with N-2 (Life Technologies), B27 Supplement without vitamin A (Life Technologies), and Glutamax. After 36 hours, media was changed to Advanced DMEM with 2% FBS, Glutamax, 5 nM GSK-3 inhibitor XV and 50 ng/mL E. coli-derived Activin A (Peprotech) for 24 hours to produce mesendoderm. For endoderm differentiation, cells were then fed with Advanced DMEM with 2% FBS, Glutamax, 50 ng/mL Activin A and 1 µM Dorsomorphin (Sigma) for 48 hours. For intestinal endoderm differentiation, cells at the endoderm stage were fed for 24 hours with Advanced DMEM with B-27 supplement without vitamin A, Glutamax, and 100 nM GSK-3 inhibitor XV.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were fixed with 1% formaldehyde for 15 minutes at room temperature. Pellets containing ~40 x10^6 cells were flash frozen and stored at -80 oC. Cells were thawed on ice, resuspended in 5ml of Lysis Buffer A and incubated for 10 minutes at 4 oC in a rotating platform. Samples were spun down for 5 minutes at 1,350g, resuspended in 5ml Lysis Buffer B and incubated for 10 minutes at 4 oC in a rotating platform. Samples were spun down for 5 minutes at 1,350g, resuspended in 3ml of Sonication Buffer (SB). Nuclear extracts were sonicated using a Misonix 3000 model sonicator to sheer cross-linked DNA to an average fragment size of approximately 500bp. Sonicated chromatin was incubated for 16 hours at 4C with Protein-G beads (Invitrogen) conjugated with either rabbit anti-V5 (Abcam) or anti-FLAG (Sigma). After incubation and with the aid of a magnetic device, beads were washed once with SB+500nM NaCl, once with LiCl Wash Buffer and 1ml of TE. Then, beads were centrifugated at 950g for 3 min and remove residual TE with a pipette. 210ul of Elution Buffer was added to the beads followed by incubation at 65 oC for 45 minutes with a brief pulse of vortex every 10 minutes. 200ul of supernatant was removed after a 1 minute centrifugation at 16,000g. The crosslink was reversed by 16 hours incubation at 65 oC. RNA was digested by the addition of 200ul of TE and RNAseA (Sigma) at a final concentration of 0.2mg/ml and incubated for 2 hours at 37C. Protein was digested by the addition of Proteinase K (0.2 mg/ml final, Invitrogen) supplemented with CaCl2 followed by a 30 minutes incubation at 55 oC. DNA was extracted with phenol:chloroform:isoamyl alcohol (25:24:1) and then recovered with an ethanol precipitation with glycogens as carrier. The pellets were suspended in 70ul of water. Purified DNA fragments were processed according to the Illumina/Solexa library protocol and sequenced using a Genome Analyzer II (Illumina, http://www.illumina.com/pages.ilmn?ID=252).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer II |
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| Description |
Sequenced input
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| Data processing |
Basecalling: Performed with CASAVA v1.7
Alignment: Sequence reads were aligned to the mouse genome (version mm9) using Bowtie (Langmead, et al. Genome Biology 2009) version 0.12.7 with options "-q --best --strata -m 1 -p 4 --chunkmbs 1024". Only uniquely mapping reads were analyzed further. All alignment data is provided here in BAM format.
BIGWIG: WIG files were generated using custom software to summarize read counts overlapping genomic regions. All reads were artificially extended out to 200bp in length. A bin size of 20bp was used for calculating overlapping read counts. Read counts per starting base pair were limited to 1 in full-length Cdx2 replicates 3 & 4, and the truncated Cdx2 experiment, due to lower library complexity than sequencing depth. WIG files were converted to bigWig using wigToBigWig (UCSC).
Genome_build: mm9
Supplementary_files_format_and_content: Alignment files are BAM format output from Bowtie; bigwig files are generated from aligned reads as described in the data processing steps.
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| Submission date |
Mar 26, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Shaun Mahony |
| E-mail(s) |
mahony@psu.edu
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| Phone |
814-865-3008
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| Organization name |
Penn State University
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| Department |
Biochemistry & Molecular Biology
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| Lab |
Shaun Mahony
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| Street address |
404 South Frear Bldg
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| City |
University Park |
| State/province |
PA |
| ZIP/Postal code |
16802 |
| Country |
USA |
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| Platform ID |
GPL9250 |
| Series (1) |
| GSE39435 |
Induced Cdx2 binding in embryonic stem cells and endodermal cells |
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| Relations |
| BioSample |
SAMN02708925 |
| SRA |
SRX501738 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1357919_Input_Endoderm.rep1.bw |
56.2 Mb |
(ftp)(http) |
BW |
| GSM1357919_Input_Endoderm.rep2.bw |
191.5 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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