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| Status |
Public on Sep 06, 2020 |
| Title |
ATACseq in EB-Day4[D0+RA+SAG][D2+48hDox(iFlag.Hoxc10)] replicate 1 |
| Sample type |
SRA |
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| Source name |
ES-derived embryoid bodies+4D RA+SAG with induced transcription factors
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| Organism |
Mus musculus |
| Characteristics |
strain genotype/variation: Ainv15(iFlag.Hoxc10)
treatment: EB + 4 Days RA + SAG, D2+48hours Dox
cell type: Embryoid Bodies
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| Treatment protocol |
Embryoid bodies (EBs) were obtained by plating trypsinized (Gibco) mESCs in AK medium (Advanced DMEM/F12:Neurobasal (1:1) medium (Gibco), 7% KnockOut SR (vol/vol) (Gibco), 2 mM L-glutamine, 0.1 mM ß-mercaptoethanol and penicillin–streptomycin (Gibco),) at 37 °C, 5% CO2 (day -2). On day 0, EBs were split 1:2 and AK medium was replenished and supplemented with 1 μM all-trans retinoic acid and 0.5 μM smoothened agonist (SAG) (Millipore, 566660). TF induction was performed by adding 3 μg/mL of Doxycycline (Sigma, D9891) on day 2. For ATAC-seq experiments, 3.5x10^5 mESCs were plated in 100mm suspension dishes (Corning).
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| Growth protocol |
mESC lines were cultured in 2-inhibitors based medium (Advanced DMEM/F12:Neurobasal (1:1) medium (Gibco), supplemented with 2.5% ESC-grade fetal bovine serum (vol/vol, Corning), N2 (Gibco), B27 (Gibco), 2mM L-glutamine (Gibco), 0.1 mM β-mercaptoethanol (Gibco), 1000 U/mL leukemia inhibitory factor (Millipore), 3μM CHIR (BioVision) and 1 μM PD0325901 (Sigma)) on 0.1% gelatin (Millipore) coated plates at 37 °C, 8% CO2.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were collected prior to TF induction (day 2 of RA/SAG differentiation) and 48h after Doxycycline (Dox) treatment. 50,000 cells were aliquoted and washed twice in 1 × PBS (cold). Cell pellets were resuspended in 10 mM Tris pH 7.4, 10 mM NaCl, 3 mM MgCl2, and freshly added 0.1% NP-40 (vol/vol), and centrifuged at 4 °C. Pellets were resuspended in 25 µL of 2 × TD buffer, 2.5 µL TDE1 (Nextera DNA sample preparation kit, FC-121–1030) and 22.5 µL of water and incubated at 37 °C for 30 min. The sample was purified using the MinElute PCR purification kit (Qiagen, 28004).
A quantitative PCR reaction with 1 × SYBR Green (Invitrogen), custom-designed primers and 2 × NEB MasterMix (New England Labs, M0541) was performed to determine the optimal number of PCR cycles (one-third of the maximum measured fluorescence) (Buenrostro, Giresi, Zaba, Chang, & Greenleaf, 2013). PCR enrichment of the library was performed with custom-designed primers and 2 × NEB MasterMix (New England Labs, M0541). The libraries were purified using the MinElute PCR purification kit. High Sensitivity DNA ScreenTape (Agilent, 5067-5584) was used to verify the fragment length distribution of the library. Library quantification was performed using the KAPA library amplification kit on the 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 |
HLVVFBGX5_n01_mb82
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| Data processing |
Basecall conversion to fastq files was performed using Picard v 2.8.2
Alignment: Paired-end ATAC-seq reads were mapped to the mouse mm10 genome using Bowtie 2.2.2 (Langmead et al., 2009).
Domain Calls: Genome-wide ATAC-seq derived accessible domains were called using DomainFinder in the SeqCode project (https://github.com/seqcode/seqcode-core/blob/master/src/org/seqcode/projects/seed/DomainFinder.java).
Genome_build: mm10
Supplementary_files_format_and_content: BED: Bed files with start and end co-ordinates corresponding to ATAC-seq domain boundaries
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| Submission date |
Dec 19, 2019 |
| Last update date |
Nov 17, 2020 |
| 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) |
| GSE142375 |
Diversification of posterior Hox patterning by graded ability to engage inaccessible chromatin [ATAC-seq] |
| GSE142379 |
Diversification of posterior Hox patterning by graded ability to engage inaccessible chromatin |
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| Relations |
| BioSample |
SAMN13635408 |
| SRA |
SRX7416010 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4226432_domains_EB_DO+RA+SAG_Day4_iHoxc10.bed.gz |
500.6 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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