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| Status |
Public on Feb 19, 2016 |
| Title |
GMP_RNAseq_bio1 |
| Sample type |
SRA |
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| Source name |
GMP_total
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| Organism |
Mus musculus |
| Characteristics |
strain: C57/Bl6
tissue: bone marrow
cell type: GMP
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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 |
polyA RNA |
| Extraction protocol |
RNA isolation was performed with the miRNeasy mini kit (Qiagen) following manufacturers instruction. To remove the feeders, cells were pre-plated for 30 minutes before RNA processing. RNA was eluted from the columns using RNase-free water and quantified by Nanodrop. cDNA was produced with the High Capacity RNA-to-cDNA kit (Applied Biosystem).
Libraries were prepared using the SMARTer Universal Low Input RNA Kit (Clontech Laboratories) according to the manufacturer's protocol.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
unstranded_RNAseq
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| Data processing |
reads were mapped using STAR5 with the Ensembl gene annotation gtf (GRCm38.78) (parameters: -sjdbOverhang 100 -outFilterType BySJout -alignSJoverhangMin 8 -alignSJDBoverhangMin 1 -outFilterMultimapNmax 1 -outFilterMismatchNmax 999 -outFilterMismatchNoverLmax 0.06 -alignIntronMin 20 -alignIntronMax 1000000 -alignMatesGapMax 1000000)
bigwig tracks were obtained with STAR
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)
Samples scaling and statistical analysis were performed using the Rpackage DESeq2 (1.6.3)
Genome_build: mm10
Supplementary_files_format_and_content: bigwig, track of read signal
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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) |
| GSE71217 |
How C/EBPa creates an elite cell state for reprogramming to pluripotency [RNAseq] |
| GSE71218 |
How C/EBPa creates an elite cell state for reprogramming to pluripotency |
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| Relations |
| BioSample |
SAMN03893482 |
| SRA |
SRX1115769 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1830149_GrafLab_GMP_RNAseq_bio1_150119_Signal.Unique.str1.out.bw |
481.2 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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