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Sample GSM4226430 Query DataSets for GSM4226430
Status Public on Sep 06, 2020
Title ATACseq in EB-Day4[D0+RA+SAG][D2+48hDox(iFlag.Hoxc9)] replicate 1
Sample type SRA
 
Source name ES-derived embryoid bodies+4D RA+SAG with induced transcription factors
Organism Mus musculus
Characteristics strain genotype/variation: Ainv15(iFlag.Hoxc9)
treatment: EB + 4 Days RA + SAG, D2+48hours Dox
cell type: Embryoid Bodies
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).
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.
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.
 
Library strategy ATAC-seq
Library source genomic
Library selection other
Instrument model Illumina NextSeq 500
 
Description HLVVFBGX5_n01_mb81
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
 
Submission date Dec 19, 2019
Last update date Nov 17, 2020
Contact name Shaun Mahony
E-mail(s) mahony@psu.edu
Phone 814-865-3008
Organization name Penn State University
Department Biochemistry & Molecular Biology
Lab Shaun Mahony
Street address 404 South Frear Bldg
City University Park
State/province PA
ZIP/Postal code 16802
Country USA
 
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
Relations
BioSample SAMN13635410
SRA SRX7416008

Supplementary file Size Download File type/resource
GSM4226430_domains_EB_DO+RA+SAG_Day4_iHoxc9.bed.gz 553.8 Kb (ftp)(http) BED
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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