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| Status |
Public on Jun 15, 2013 |
| Title |
H3K4me1_MES_Rep_3 |
| Sample type |
SRA |
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| Source name |
MES stage cultures, H3K4me1
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| Organism |
Mus musculus |
| Characteristics |
strain: 129/Ola
time point: Differentiation Day 4
cell type: mesodermal cells (MES)
antibody: H3K4me1 (Abcam ab8895)
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| Growth protocol |
Embryonic stem cells were differentiated as EBs for two days in serum-free media (3 parts IMDM (Cellgro #15-016-CV): 1 part Ham’s F12 (Cellgro #10-080-CV), 0.05% BSA, 2 mM GlutaMax (GIBCO), B27 supplement (GIBCO #12587010), N2 supplement (GIBCO #17502048)) supplemented with 50 mg/ml ascorbic acid and 4.5 3 10^4 M monothioglycerol. Embryoid bodies were dissociated and reaggregated for 40 hr in the presence of 5 ng/mL human VEGF (R&D #293-VE) and human Activin A (R&D #338-AC) and human BMP4 (R&D #314-BP) at concentrations empirically determined depending on lot. EBs were dissociated and plated at 470,000 cells/cm2 in StemPro-34 (GIBCO #10639011) supplemented with 5 ng/mL VEGF, 10 ng/mL human basic FGF (R&D #233-FB) and 25 ng/mL FGF10 (R&D #345- FG).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin immunoprecipitation of histone modifications were performed according to the Young lab protocol (Lee et al., 2006) with minor modification. Briefly, frozen pellets of cross-linked cells (10 3 106) were thawed in cold lysis buffer 1 (50 mM HEPES-KOH, pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100, 1 3 protease inhibitors) and gently rocked at 4 C for 10 min in 14 ml conical tubes. Cells were pelleted at 1350 x g at 4 C in a clinical centrifuge and resuspended in cold lysis buffer 2 (10 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1 3 protease inhibitors) and gently rocked at 4 C for 10 min in 14 ml conical tubes. Cells were pelleted at 1350 x g at 4C in a table top centrifuge and resuspended in 2 ml cold lysis buffer 3 (10 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Na-Deoxycholate, 0.5% N-lauroylsarcosine, 1 3 protease inhibitors) and sonicated to 200-600 bp fragments using a Diagenode Bioruptor (3 3 10 min cycles, 30 s ON / 30 s OFF at 4 C). Soni- cated lysates were cleared by pelleting insoluble material at 20,000 x g at 4 C followed by incubation with antibody bound Protein A/G magnetic beads (2.5 mg Ab / 50uL beads / IP) in 1 ml of 0.5% BSA/PBS overnight at 4 C. Magnetic beads were washed 3 times with block (0.5% BSA/PBS), incubated for approximately 4 hr at 4 C with antibody in block and then washed 3 times with block prior to addition of cleared cell lysates. Immunoprecipitated material was washed five times with cold wash buffer (RIPA: 50 mM HEPES-KOH, pKa 7.55, 500 mM LiCl, 1 mM EDTA, 1.0% NP-40, 0.7% Na-Deoxycholate) and one time with TE plus NaCl, followed by elution and uncrosslinking in 210 uL of 1% SDS in TE overnight at 65 C. 200 uL of uncrosslinked material was treated with RNase A for 2 hr, proteinase K for 2 hr and extracted 2 times with phenol chloroform isoamyl alcohol, followed by ethanol precipitation with a glycogen coprecipitant, 80% ethanol wash and final resuspension in TE.
Illumina sequencing libraries were generated (Schmidt et al., 2009), with minor modi- fication. Briefly, 5-50 ng of immunoprecipitated nucleic acid was end repaired, a-tailed and ligated to Illumina single end adapters using an NEB Next kit (New England Biolabs). 200-300 bp adaptor ligated nucleic acid was gel purified and enriched via 19 cycles of PCR amplification with Phusion High-Fidelity DNA Polymerase, followed by sequencing on an Illumina HiSeq 2000 system
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
Short reads were aligned to the mm9 mouse genome using Bowtie (2 bp mismatch)
Sequences were extended +200 bp for histone marks and RNAPser5 and allocated in 25-bp bins. Extended sequences were also processed into Wiggle format for visualization.
Biological replicate whole cell extracts were sequenced for each time point and combined for use as background model. A Poissonian model was used to determine statistically enriched bins with a p value threshold set at 1 X 10^9 as described previously (Marson et al., 2008) and a 5-fold enrichment over input cutoff.
Genome_build: mm9
Supplementary_files_format_and_content: .wig files,representing read density across the genome, were generated after extending aligned sequences by 200 nt.
Supplementary_files_format_and_content: *_ENRICHED_REGIONS.txt files are encoded in bed format and contain genomic regions corresponding to enriched ChIP-Seq regions.
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| Submission date |
Jun 14, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Laurie A Boyer |
| E-mail(s) |
lboyer@mit.edu
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| Phone |
617 324-3335
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| Organization name |
Massachusetts Institute of Technology
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| Department |
Biology
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| Lab |
Boyer
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| Street address |
77 Massachusetts Avenue
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| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02139 |
| Country |
USA |
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| Platform ID |
GPL13112 |
| Series (2) |
| GSE47949 |
ChIP-seq analysis of histone modifications and RNA polymerase II at 4 stages of directed cardiac differentiation of mouse embryonic stem cells |
| GSE47950 |
Dynamic and Coordinated Epigenetic Regulation of Developmental Transitions in the Cardiac Lineage |
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| Relations |
| BioSample |
SAMN02203973 |
| SRA |
SRX305895 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1163089_H3K4me1_MES_Rep_3_JW-H68.wig.gz |
9.5 Mb |
(ftp)(http) |
WIG |
| GSM1163089_H3K4me1_MES_Rep_3_JW-H68_ENRICHED_REGIONS.txt.gz |
871.7 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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