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| Status |
Public on Dec 31, 2015 |
| Title |
flag_Oct4_ChipSeq_day5 |
| Sample type |
SRA |
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| Source name |
reprogramming intermediate after 5days of induction
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| Organism |
Mus musculus |
| Characteristics |
cell type: reprogramming intermediate
genotype: Oct4-GFP/ Rosa26-M2rtTA transgenic background
chip-antibody: anti-Flag-M2, Sigma(F1804)
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| Growth protocol |
The 2nd mouse embyronic fibroblast were derived from E13.5 tetraploid complementated all-iPS embryos and cultured with Feeder Medium. On passages 4, MEFs were plated on gelatin-coated dishes at appropriate density and the culture medium was changed to ES Medium supplemented with 1µg/ml Doxcycline and 50µg/ml Vitamin C to induce iPS cells. After 15days of induction, majority of cells were reprogrammed into Oct4-GFP+ ES-like colonies and we withrew Doxcycline and Vc from the medium. 3days later, the remaining dox-indenpent iPS cells were collected as day18 sample. The 2nd iPS cell line was established and maintained in ES medium supplemented with 2i(Gsk3i+Meki) with/without feeder. Feeder Medium contains DMEM supplemented with 10% (v/v) fetal bovine serum and 1 mM L-glutamine; ES Medium contains DMEM supplemented with 15% (v/v) fetal bovine serum , 1 mM L-glutamine, 0.1 mM mercaptoethanol, 1% nonessential amino acid stock,1% penicillin/streptomycin stock,1% nucleosides stock and 1000 U/ml LIF.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Reprogramming cells at different time point were trypsinized and washed twice with PBS. Cells were resuspended in PBS at a density of 107/ml and then chemically crosslinked by the addition of final 1% formaldehyde for 10 min at room temperature with gently rotation. Crosslinking was stopped by addition of glycine to a final concentration of 125 mM for 5 min at room temperature. Cells were washed twice with cold PBS and lysed in Lysis Buffer(10 mM Tris-HCl, pH 7.0, 10 mM NaCl, 3 mM MgCl2, 0.5% NP40; freshly added complete proteinase inhibitor cocktail (Roche)) at 4℃ for 15 min. Nuclei was collected by centrifugation and washed again with Lysis Buffer. Pellet was resuspended in ChIP Buffer I (300 mM NaCl, 3 mM EDTA, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 100 μg /ml BSA, 50 mM Tris, pH 8.0; freshly added proteinase inhibitor cocktail) and sheared by sonication to reduce the average DNA fragment size to 200-500 bp for analysis by q-PCR or 100-300 bp for analysis by sequencing. Antibodies were pre-coupled to protein G dynabeads. Generally, we used 10 μl protein G Dynabeads for 1 μg antibody. For transcription factor ChIP, we generally used 3 μg antibody for 30 μg chromatin per reaction; for histone ChIP, we used 1-2 μg antibody for 10-20 μg chromatin per reaction. After sonication, 1/10 of sheared chromatin was saved as input. The remaining chromatin was incubated with antibody-coupled dynabeads at 4℃ overnight. Beads were washed twice by ChIP Buffer I, four times by ChIP buffer II (100 mM Tris, pH 8.0, 500 mM LiCl, 1% NP40, 1% sodium deoxycholate) at 4℃ for 5 min with gently rotation. Beads were finally washed once by TE (10 mM Tris-HCl pH 8.0, 0.1 mM Na2EDTA) and transferred to a new tube for elution. Bound complexes were eluted twice in 100 μl Elution Buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 0.1% SDS; 0.25 mg/mL proteinase K added freshly) at 55℃ for 1 hr with shaking. The eluted chromatin was further reverse-crosslinked by overnight incubation at 65℃.Input chromatin was also treated to reverse crosslinking. Immunoprecipitated DNA and input DNA was extracted by phenol: chloroform: isoamyl alcohol (25:24:1, pH 8.0) and precipitated by ethonal with 100 μg /ml LPA as co-precipitant.
For the following sequencing analysis, 10-100 ng DNA mixed from two or three independent ChIP reactions was used to prepare ChIP-seq library following the NEB protocol with minor modifications. Briefly, DNA was end-repaired, dA-tailed and ligated with NEBNext adaptors. Following USER enzyme treatment, DNA was purified and size selected with 0.8X\0.4X AMpureXP beads (Beckman). Selected DNA was PCR amplified using Phusion (NEB) or Q5 (NEB) DNA polymerase for 12-14 cycles. PCR primers were from NEBNext Multiplex Oligos for Illumina kit (NEB). Different samples were tagged with different indexes and 3-4 ChIP-libraries were mixed as one lane sample in HiSeq 2000/HiSeq 2500.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
exogenous Oct4 binding
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| Data processing |
Basecalls performed using CASAVA version 1.4
ChIP-seq reads were aligned to the mm9 genome assembly using bowtie version 0.12.7 with the following configurations:bowtie -v 2 -m1
Only these uniquely mapped reads were kept with the following command: samtools view -b -q 255 data.bam >uniquely_data.bam
peaks were called using macs2 version 2.1.0 with the default parameters plus --broad: macs2 -t treatment.bam -c input.bam --broad. H3K4me1, H3K27ac, H3K4me3, H3K27me3 and RNAPII peaks were called using macs version 1.4.1 with the following setting: macs14 -t treatment.bam -c input.bam --keep-dup auto -w -S --space 30 --fe-step=10.
Genome_build: mm9
Supplementary_files_format_and_content: broadPeak, bed
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| Submission date |
Apr 01, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Min Li |
| E-mail(s) |
limin18.love@163.com
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| Organization name |
Tongji University
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| Street address |
1239 Siping Road, Yangpu District
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| City |
Shanghai |
| ZIP/Postal code |
200092 |
| Country |
China |
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| Platform ID |
GPL13112 |
| Series (1) |
| GSE67520 |
Oct4 binding and Histone modification profiling during OSKM-mediated 2nd reprogramming |
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| Relations |
| BioSample |
SAMN03458347 |
| SRA |
SRX977368 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1648619_Flag_d5_peaks.broadPeak.gz |
2.9 Mb |
(ftp)(http) |
BROADPEAK |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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