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| Status |
Public on Feb 19, 2016 |
| Title |
Bcell_Cebpa18h_OSKM1d_ChIPseq_input |
| Sample type |
SRA |
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| Source name |
Bcell+Cebpa18h+OSKM1d
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| Organism |
Mus musculus |
| Characteristics |
strain: C57/Bl6
tissue: bone marrow
cell type: B cell
antibody: none (input)
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| Treatment protocol |
For the reprogramming experiments B cells were exposed for 18h to 100nM of β-Estradiol (E2) in gelatinized plates seeded with a feeder layer of the MEFs in RPMI medium. After E2 washout, the cultures were switched to serum-free N2B27 medium supplemented with 2μg/ml of doxycycline, IL-4 10ng/ml, IL-7 10ng/ml and IL-15 2ng/ml. B cells were seeded at a density of 500 cells/cm2 in 12 well plates.
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| Growth protocol |
ESCs (E14TG2) were cultured on gelatinized plates in N2B27 media (50 %DMEM/F12, 50% Neurobasal medium, N2 (500X), B27 (1000X)) supplemented with small-molecule inhibitors PD (1 μM, PD0325901), CHIR (3 μM, CHIR99021) and LIF. CD19+ pre-B cells were obtained from bone marrow with monoclonal antibody to CD19 (BD Pharmingen) using MACS sorting (Miltenyi Biotech). The purity of the sorted cell fractions was confirmed by FACS using an LSR2 machine (BD). After isolation, B cells were grown in RPMI medium supplemented with 10% FBS and 10ng/ml IL-7 (Peprotech). All medium contain 100 X L-glutamine, 100X penicillin/streptomycin, 100X nonessential amino acids, 1000X β-mercaptoethanol. All reagents are from Life Technologies unless otherwise specified. Lin- c-Kit+ Sca-1- CD16+/CD32+ CD34+ GMP cells were isolated by FACS sorting using a BD INFLUX sorting machine and culture in STEMSPAN medium (Stemcell technologies) supplemented with 100ng/ml SCF, 50ng/ml IL3, 50ng/ml Flt3L and 50ng/ml mTPO (all from Peprotech). The antibodies used from GMPs isolation were from BD Biosciences.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP was performed as described in Jakobsen et al, BMC genomics, 16, 46 (2015)
Amplification of 2 ng ChIP DNA was essentially performed as described by the manufacturer (NEB, cat#E6240S), with the use of precast 2% SYBR agarose gels (Invitrogen, cat#G5218-02) and excision of band size 175–400 bp. Key modifications consisted of a 30 minute ligation step, 30 minute gel solubilization at 37°C of excised gel fragments, and a prolonged, double run-through elution step (each three minutes) with preheated (55°C) elution buffer for all column purifications (Qiagen, cats#28104,28704,28004) to ensure robust recovery.
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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 |
reads were mapped using STAR (parameters: -outFilterMultimapNmax 1 -outFilterMismatchNmax 999 -outFilterMismatchNoverLmax 0.1 -alignIntronMax 1 -alignEndsType EndToEnd).
Duplicates reads were removed using picard
For quantitative analysis, reads were counted on genes exon using the Rpackage Rsubread (1.16.1)(parameters: GTF.featureType="exon", GTF.attrType="gene_id", isPairedEnd=T, nthreads=12, strandSpecific=0)
bigwig tracks were made using DeepTools BamCompare to subtract the input from the ChIP (parameters: -scaleFactorsMethod SES -ratio subtract -normalizeUsingRPKM -fragmentLength 200 -binSize 1)
Genome_build: mm10
Supplementary_files_format_and_content: bigwig, track of fragment signal (elongated to 200b)
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| Submission date |
Jul 22, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
samuel Collombet |
| E-mail(s) |
samuelcollombet@gmail.com
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| Organization name |
Ecole Normale Superieure
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| Department |
Biology
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| Lab |
Thieffry
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| Street address |
46 rue d'Ulm
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| City |
Paris |
| ZIP/Postal code |
75005 |
| Country |
France |
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| Platform ID |
GPL13112 |
| Series (2) |
| GSE71215 |
How C/EBPa creates an elite cell state for reprogramming to pluripotency [ChIPseq] |
| GSE71218 |
How C/EBPa creates an elite cell state for reprogramming to pluripotency |
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| Relations |
| BioSample |
SAMN03893448 |
| SRA |
SRX1115740 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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