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Sample GSM1830129 Query DataSets for GSM1830129
Status Public on Feb 19, 2016
Title Bcell_ChIPseq_Cebpa
Sample type SRA
Source name Bcell
Organism Mus musculus
Characteristics strain: C57/Bl6
tissue: bone marrow
cell type: B cell
target: ChIP Cebpa
antibody: Santa Cruz, SC-61
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.
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.
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.
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina NextSeq 500
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)
Submission date Jul 22, 2015
Last update date May 15, 2019
Contact name samuel Collombet
Organization name Ecole Normale Superieure
Department Biology
Lab Thieffry
Street address 46 rue d'Ulm
City Paris
ZIP/Postal code 75005
Country France
Platform ID GPL19057
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
BioSample SAMN03893459
SRA SRX1115751

Supplementary file Size Download File type/resource 362.3 Mb (ftp)(http) BW
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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