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| Status |
Public on Oct 01, 2021 |
| Title |
Embryonic Stem Cells (ATAC-Seq) Replicate 1 |
| Sample type |
SRA |
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| Source name |
WiCell
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| Organism |
Homo sapiens |
| Characteristics |
cell stage: Embryonic Stem Cell
age (differentiation protocol): 0
esc line: human ESC H9
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| Treatment protocol |
ESC were untreated. Upon confluency, cells were cultured in ESC medium without FGF-Basic and Y-27632, but supplemented with Shh (100 ng/ml), Purmorphamine (2 μM), 10 μM SB431542, and 2.5 μM LDN193289. From days 5 to 8, ESC medium was gradually replaced with neuroprogenitor medium (DMEM/F-12 supplemented with 0.1 mM MEM Non-Essential Amino Acids, N-2 Supplement, 0.2 μM ascorbic acid, 0.16% glucose). At day 9, cells were switched into neuronal differentiation medium (DMEM/F-12 supplemented with 0.1 mM MEM Non-Essential Amino Acids, N-2 Supplement, B-27 Supplement minus Vitamin A, 0.2 μM ascorbic acid, 0.16% glucose containing 10 μM DAPT). On day 12, cells were collected with TrypLE Express Enzyme at 37°C for 7 min and washed twice including filtration through pre-wetted 40 μm Corning Sterile Cell Strainer. The hypothalamic progenitor cell pellet was then resuspended with neuronal differentiation medium containing 10 μM Y-27632 for plating on 148 cm2 dishes coated with Poly-L-Ornithine solution (0.01%) and laminin (4 μg/ml) at a seeding density of 16 million cells/148 cm2. After 4 hours (once the cells had completely attached to the plate), the medium was changed to neuronal differentiation medium supplemented with 10 μM DAPT. On day 15, the neuronal differentiation medium was supplemented with 20 ng/ml BDNF (Peprotech Cat # 450-02) until collection on day 27.
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| Growth protocol |
Human ESC H9 (W09; WiCell) were seeded on Matrigel plates (16 million cells/148 cm2) in DMEM KO medium supplemented with 15% KnockOut Serum Replacement, 0.1mM MEM Non-Essential Amino Acids, 2mM GlutaMAX, 0.06 mM 2-Mercaptoethanol) with FGF-Basic (20 ng/ml media), and 10 mM Rock inhibitor.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
A total of 50,000 cells were centrifuged at 550g for 5 min at 4°C. The cell pellet was washed with cold PBS and resuspended in 50 μL cold lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% NP-40/IGEPAL CA-630) and immediately centrifuged at 550 g for 10 min at 4°C. The tagmented DNA was then purified using the Qiagen MinElute kit and eluted in 10.5 μL Elution Buffer.
Ten microliters of purified tagmented DNA was PCR amplified using the Nextera Indexing Kit for 12 cycles to generate each library. The PCR reaction was subsequently purified using 1.8x AMPure XP beads, and concentrations were measured by Qubit Fluorometer. Quality of completed libraries was assessed on a Bioanalyzer 2100 high sensitivity DNA Chip. Libraries were paired-end sequenced at the Center for Spatial and Functional Genomics on the Novaseq 6000 platform (50 bp read length).
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| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
HPvsESC.DE_atacseq_edgeR.txt
HNvsESC.DE_atacseq_edgeR.txt
HypothalamusESC_atac_counts_fpkm.txt
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| Data processing |
Bases were called using Illumina bcl2fastq2 conversion v2.29
Reads were aligned using bowtie2 implimented in the ENCODE pipeline. Reads alignment by offsetting +4bp for all the reads aligned to the forward strand, and -5bp for all the reads aligned to the reverse strand
Open chromatin regions (OCRs) were called using MACS2 implimented in the ENCODE pipeline with the following parameters -p 0.01 --nomodel --shift -75 --extsize 150 -B --SPMR --keep-dup all --call-summits. The encode blacklist regions (wgEncodeDacMapabilityConsensusExcludable.bed.gz) were removed from called peaks.
Prior to differential analysis peaks were filtered to remove peaks not identified in at least half of replicates using bedtools intersect (v2.25.0). A consensus set of peaks was generated by taking the union of peaks defined in the three stages of differentiation
Read counts for the consensus set of peaks were normalized using csaw v1.8.1. OCRs with maximium median value of less than 1.2 CPM (~10-50 reads per OCR) across all replicates were filtered prior to differential analysis.
Differential analysis was performed using the glmQLFit implmented in edgeR v 3.16.5 using the normalization factors calculated with csaw. Differential OCRs were identified with thresholds of FDR < 0.05 and log2 fold change > 1.
FPKM values were calculated from reads mapped to consensus OCRs and bigWig files were generated from the duplicate filtered bam files using the R packages GenomicAlignments (v1.10.1) & rtracklayer(1.34.2)
Genome_build: hg19
Supplementary_files_format_and_content: bigwig files for each replicate as well as a tab-deliminated file containing OCR locations, raw counts and fpkm, as well as results from each differential comparison
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| Submission date |
Jun 09, 2020 |
| Last update date |
Oct 01, 2021 |
| Contact name |
Matthew C Pahl |
| E-mail(s) |
pahlm@email.chop.edu
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| Phone |
5704282303
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| Organization name |
Children's Hospital of Philadelphia
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| Department |
Center for Spatial and Functional Genomics
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| Lab |
Struan Grant and Andrew Wells
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| Street address |
3615 Civic Center Blvd
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| City |
Philadelphia |
| State/province |
PA |
| ZIP/Postal code |
19104 |
| Country |
USA |
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| Platform ID |
GPL24676 |
| Series (2) |
| GSE152090 |
Cis-regulatory architecture of human ESC-derived hypothalamic neuron differentiation aids in variant-to-gene mapping of relevant common complex traits (ATAC-Seq) |
| GSE152098 |
Cis-regulatory architecture of human ESC-derived hypothalamic neuron differentiation aids in variant-to-gene mapping of relevant common complex traits |
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| Relations |
| BioSample |
SAMN15186404 |
| SRA |
SRX8508292 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4602954_HumanESCells_rep1.bigwig |
176.3 Mb |
(ftp)(http) |
BIGWIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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