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| Status |
Public on Feb 19, 2016 |
| Title |
Bcell_OKSMd2_ATAC |
| Sample type |
SRA |
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| Source name |
Bcell+Cebpa18h+OSKM2d
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| Organism |
Mus musculus |
| Characteristics |
strain: C57/Bl6
tissue: bone marrow
cell type: B cell
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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 |
ATACseq was performed as in (Buenrostro et al, Nature methods 10, 1213-1218 (2013)) with the following modifications. 100.000 cells were washed once with 100µl PBS and resuspended in 50µl lysis buffer (10mM Tris-HCl pH 7.4, 10mM NaCl, 3mM MgCl2, 0.2% IGEPAL CA-630). The nuclei suspension was then centrifuged for 10min at 500g at 4C. The 50µl transposition reaction (25µl TD buffer, 2.5µl Tn5 Transposase and 22.5µl Nuclease Free H2O) was added to the isolated nuclei and incubated at 37C for 45min. DNA was isolated using Qiagen MinElute Kit following the manufacturer instructions.
Library amplification was done by two sequential PCR reactions (8 and 5 cycles, respectively). After the first PCR reaction the library was size selected (for fragments below 700bp) with AmpureXP beads followed by a second PCR reaction. Libraries were purified with Qiaquick PCR purification and integrity checked on a Bionalyzer before sequencing.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
Transposase accessible chromatin
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| Data processing |
Library strategy: ATAC-seq
reads were mapped using STAR (parameters: -outFilterMultimapNmax 1 -outFilterMismatchNmax 999 -outFilterMismatchNoverLmax 0.06 -alignIntronMax 1 -alignEndsType EndToEnd -alignMatesGapMax 2000, which forbids read splitting and results in a mapping similar to a classical genomic mapper, and allow a maximum of 2kb between two reads of a pair).
Duplicates reads were removed using Picard (http://picard.sourceforge.net./) (function MarkDuplicates, parameter REMOVE_DUPLICATES=true).
bigwig tracks were made using DeepTools BamCoverage (1.5.9.1) (parameters: -binSize 1 -normalizeUsingRPKM).
Genome_build: mm10
Supplementary_files_format_and_content: bigwig, track of fragments 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) |
| GSE71214 |
How C/EBPa creates an elite cell state for reprogramming to pluripotency [ATACseq] |
| GSE71218 |
How C/EBPa creates an elite cell state for reprogramming to pluripotency |
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| Relations |
| BioSample |
SAMN03893442 |
| SRA |
SRX1115733 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1830113_GrafLab_Bcell_OKSMd2_ATAC_Aligned.sortedByCoord.out.rmdup.bam.bw |
355.5 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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