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Sample GSM4226427 Query DataSets for GSM4226427
Status Public on Sep 06, 2020
Title ATACseq in EB-Day2[D0+RA+SAG]_replicate2
Sample type SRA
Source name ES-derived embryoid bodies+2D RA+SAG
Organism Mus musculus
Characteristics strain genotype/variation: Ainv15(iCre)
treatment: EB + 2 Days RA + SAG
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_mb84
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 (
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
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
BioSample SAMN13635413
SRA SRX7416005

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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