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Sample GSM1956908 Query DataSets for GSM1956908
Status Public on Feb 19, 2016
Title Bcell_4C_Klf4enhancer
Sample type SRA
 
Source name Bcell
Organism Mus musculus
Characteristics target: Klf4 enhancer
first cutter and restriction site sequence: Csp6i (GTAC)
second cutter and restriction site sequence: DpnII (GATC)
reading primer sequence: CGCTTTATGTTCTGCCAGTAC
non-reading primer sequence: TGTCACAGCCCCAGTAGTG
Treatment protocol For the reprogramming experiments B cells were exposed for 18h to 100nM of -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 0.5-1.0 million of crosslinked nuclei were digested with Csp6I followed by ligation under dilute conditions. After decrosslinking and DNA purification, samples were digested overnight with DpnII and once more ligated under dilute conditions.
Column purified DNA was directly used as input for inverse PCR using primers with Illumina adapter sequences as overhangs. Several PCR reactions were pooled, purified and sequenced.
 
Library strategy OTHER
Library source genomic
Library selection other
Instrument model Illumina HiSeq 2000
 
Data processing Library strategy: 4C-seq
The reading primer was removed from the read seqeunce using the python script present in the bioconductor package fourCseq demultiplex.py
reads were mapped using STAR (parameters: -outFilterMultimapNmax 1 -outFilterMismatchNmax 999 -outFilterMismatchNoverLmax 0.1 -alignIntronMax 1 -alignEndsType EndToEnd).
Mapped reads were pre-process using the fourCseq package, and tracks of signal were made using the R package fourCseq, applying a running mean over 5 framgments.
Genome_build: mm10
Supplementary_files_format_and_content: bigwig, track of signal
 
Submission date Nov 30, 2015
Last update date May 15, 2019
Contact name samuel Collombet
E-mail(s) samuelcollombet@gmail.com
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 GPL13112
Series (2)
GSE71218 How C/EBPa creates an elite cell state for reprogramming to pluripotency
GSE75481 How C/EBPa creates an elite cell state for reprogramming to pluripotency [4C]
Relations
BioSample SAMN04301821
SRA SRX1456146

Supplementary file Size Download File type/resource
GSM1956908_Klf4Enhancer_Bcell_2_FragCounts_RPM_rollmean5frag.bw 20.8 Mb (ftp)(http) BW
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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