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| Status |
Public on Feb 27, 2019 |
| Title |
ChIPseq Ascl1 in EB+12hrDox(iAscl1.V5) replicate 3 |
| 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.V5)
chip target: Ascl1
chip antibody: anti-Ascl1 (Abcam ab74065)
treatment: Dox induction + 12 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 ChIP-seq experiments, the seeded cell number was scaled up to 3-3.5x10^6 cells in 245 mm x 245 mm square 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 |
ChIP-seq: Cells were collected at 12 hours and 48 hours after TF induction and fixed with 1mM DSG (ProtoChem) followed by 1% FA (vol/vol) each for 15 min at room temperature. Pellets containing 25-30x10^6 cells were aliquoted and flash-frozen at -80°C. Cell aliquots were thawed on ice and resuspended in 5 ml of cold lysis buffer 1 (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol (vol/vol), 0.5% Igepal (vol/vol), 0.25% Triton X-100 (vol/vol)) with 1X protease inhibitors (Roche, 11697498001) and incubated at 4°C on a rotator. The samples were centrifuged for 5 min at 2300 rpm at 4°C and resuspended in 5ml of cold lysis buffer 2 (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol (vol/vol), 0.5% Igepal (vol/vol), 0.25% Triton X-100 (vol/vol)) with 1X protease inhibitors and incubated 10 min at 4°C on a rotator. Nuclear extracts were centrifuged for 5 min at 2300 rpm resuspended in cold 3 ml of sonication buffer (50mM HEPES pH 7.5, 140 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 0.1% sodium deoxycholate (wt/vol), 0.1% SDS (wt/vol). Sonication of the nuclear extracts was performed on ice with Branson 450 digital sonifier (Marshall Scientific, B450CC) at 20% amplitude, 18 cycles of 30s ON/60s OFF, to sheer crosslinked chromatin into average size of approximately 300 bp. Immunoprecipitation of the sonicated chromatin with Dynabeads protein-G (Thermo Fisher) conjugated antibodies were done overnight (16 hr) at 4°C on a rotator. 5 ug of following antibodies were used for immunoprecipitation.
In-house library preparation kit was used for ChIP-seq: 24ul of ChIP DNA (1:100 dilution of input DNA) was used to prepare lllumina DNA sequencing libraries. Bioo Scientific multiplexed adapters were ligated after end repair and A-tailing, and unligated adapters were removed by purification using Agencourt AmpureXP beads (Beckman Coulter). Adapter-ligated DNA was amplified by PCR using TruSeq primers (Sigma). DNA libraries between 300 and 500 bp in size were purified from agarose gel purified using Qiagen minElute columns. 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 |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
Ascl1_Ngn2_06042017_EB+12hr_iA_Ascl1.events.txt
Ascl1_Ngn2_06042017_EB+12hr_iA_Ascl1.bed
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| Data processing |
Basecall conversion to fastq files was performed using Picard.
Alignment: fastq files were aligned to the mouse genome (version mm10) using Bowtie (1.0.1) (Langmead et al., 2009) with options “-q --best --strata -m 1 --chunkmbs 1024”. Only uniquely mapped reads were considered for further analysis.
Binding event detection: MultiGPS was used to define transcription factor DNA binding events (Mahony et al., 2014). A q-value cutoff of 0.01 (assessed using binomial tests and Benjamini-Hochberg multiple hypothesis test correction), was used to call statistically significant binding events. Differential binding analysis between timepoints (12hr vs 48hr) or factor inductions (iAscl1 vs iNeurog2) was also performed using MultiGPS, which calls EdgeR (Robinson et al., 2009) internally. Differentially bound sites are defined as those that display significantly greater read enrichment levels (minimum 1.5-fold, q-value < 0.05) as determined by EdgeR’s negative binomial generalized linear models applied to MultiGPS’ per-replicate count data (TMM normalized).
Genome_build: mm10
Supplementary_files_format_and_content: MultiGPS: MultiGPS output files
Supplementary_files_format_and_content: BED: Conversion of MultiGPS output files to BED format, centered on the MultiGPS binding event location
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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) |
| GSE114172 |
Molecular basis of neuronal subtype bias introduced by proneural factors Ascl1 and Neurog2 (ChIP-seq) |
| GSE114176 |
Molecular basis of neuronal subtype bias introduced by proneural factors Ascl1 and Neurog2 |
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| Relations |
| BioSample |
SAMN09091468 |
| SRA |
SRX4054006 |
| 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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