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| Status |
Public on Feb 27, 2019 |
| Title |
ATACseq in EB+48hrDox(iAscl1[bHLH:Ngn2].HA) replicate 1 |
| Sample type |
SRA |
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| Source name |
ES-derived embryoid bodies with induced transcription factors
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| Organism |
Mus musculus |
| Characteristics |
strain: Ainv15(iAscl1[bHLH:Ngn2].HA)
treatment: Dox induction + 48 hours
cell type: Embryoid Bodies
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| Treatment protocol |
After two days, the EBs were passaged 1:2 and expression of the transgenes was induced by 3 ug/ml Doxycycline (Sigma D9891) to the AK medium. For ATAC-seq experiments, 2-3x10^5 cells were plated in each 100 mm untreated dishes (Corning).
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| Growth protocol |
The inducible mESCs were grown in 2i (2-inhibitors) based medium (Advanced DMEM/F12: Neurobasal (1:1) Medium (GIBCO), supplemented with 2.5% mESC-grade fetal bovine serum (vol/vol, Corning), N2 (GIBCO), B27 (GIBCO), 2mM L-glutamine (GIBCO), 0.1 mM ß-mercaptoethanol (GIBCO), 1000 U/ml leukemia inhibitory factor (Millipore), 3mM CHIR (BioVision) and 1 mM PD0325901 (Sigma) on 0.1% gelatin (Milipore) coated plates at 37°C, 8% CO2. To obtain embryoid bodies (EBs), 60-70% confluent mESCs were dissociated by TrpLE (Gibco) and plated in AK medium (Advanced DMEM/F12: Neurobasal (1:1) Medium, 10% Knockout SR (vol/vol) (GIBCO), Pen/Strep (GIBCO), 2mM L-glutamine and 0.1mM ß-mercaptoethanol) on untreated plates for two days (day -2) at at 37°C, 8% CO2.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
ATAC-seq: 50.000 cells were harvested and washed twice in cold 1X PBS. Cells were resuspended in 10mM Tris pH 7.4, 10mM NaCl, 3mM MgCl2, and 0.1% NP-40 and centrifuged immediately at 4°C. The pellet was resuspended in 25 ul of 2x TD buffer, 2.5 ul TDE1 (Nextera DNA sample preparation kit, FC-121-1030) followed by incubation for 30 min at 37C. The reaction was then cleaned by Min-elute PCR purification kit (Qiagen, 28004). The optimal number of PCR cycles were determined to be the 1/3 of the maximum fluorescence measured by qPCR reaction with 1X SYBR Green (Invitrogen), custom designed primers (Buenrostro et al., 2013) and 2X NEB MasterMix (New England Labs, M0541). Following PCR enrichment, the library was cleaned with min-elute PCR kit and quantified using Qubit (Life Technologies, Q32854).
Nextera DNA sample preparation kit, FC-121-1030 was used for ATAC-seq. The fragment length distribution of the library was determined by Agilent High Sensitivity DNA D1000 Screentape (5067- 5585) system and the final quantification of the library before pooling was done with KAPA library amplification kit (Roche Lightcycler 480).
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| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
iAscl1[bHLH-Neurog2]_48h.atac-domains.p0.05.bed
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| Data processing |
Basecall conversion to fastq files was performed using Picard.
Alignment: ATAC-seq data was mapped to the mouse genome (version mm10) using bowtie2-2.2.2 (Robinson et al., 2009) using “-q --very-sensitive” options.
Domain-finding: Enriched domains were identified using the DomainFinder module in SeqCode: (https://github.com/seqcode/seqcode-core/blob/master/src/org/seqcode/projects/seed/DomainFinder.java). Briefly, contiguous 50bp genomic bins with significantly higher read enrichment compared to normalized input were identified (binomial test, p-value < 0.05). Further, contiguous blocks within 200bp were joined together to call enriched domains.
Genome_build: mm10
Supplementary_files_format_and_content: BED: Domains displaying significant enrichment of ATAC-seq reads
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| Submission date |
May 08, 2018 |
| Last update date |
Feb 27, 2019 |
| Contact name |
Shaun Mahony |
| E-mail(s) |
mahony@psu.edu
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| Phone |
814-865-3008
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| Organization name |
Penn State University
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| Department |
Biochemistry & Molecular Biology
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| Lab |
Shaun Mahony
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| Street address |
404 South Frear Bldg
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| City |
University Park |
| State/province |
PA |
| ZIP/Postal code |
16802 |
| Country |
USA |
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| Platform ID |
GPL19057 |
| Series (2) |
| GSE114171 |
Molecular basis of neuronal subtype bias introduced by proneural factors Ascl1 and Neurog2 (ATAC-seq) |
| GSE114176 |
Molecular basis of neuronal subtype bias introduced by proneural factors Ascl1 and Neurog2 |
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| Relations |
| BioSample |
SAMN09091378 |
| SRA |
SRX4053982 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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