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| Status |
Public on Oct 01, 2021 |
| Title |
Embryonic Stem Cell dervived Hypothalamic Progenitor-like Cells(Capture C) Replicate 3 |
| Sample type |
SRA |
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| Source name |
WiCell
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| Organism |
Homo sapiens |
| Characteristics |
cell type: Hypothalamic Progenitor
age (differentiation protocol): 12
esc line: human ESC H9
restriction enzyme: DpnII
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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 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 |
For each library, 107 fixed cells were thawed at 37ºC, followed by centrifugation at RT for 5 mins at 14,000 rpm. The cell pellet was resuspended in 1 mL of dH2O supplemented with 5 μL 200X protease inhibitor cocktail, incubated on ice for 10 mins, then centrifuged. The cell pellet was resuspended to a total volume of 650 μL in dH2O.
Both samples underwent a pre-digestion incubation with the addition of 0.3% SDS, 1x NEBuffer DpnII, and dH2O for 1hr at 37ºC in a Thermomixer shaking at 1,000rpm. A 1.7% solution of Triton X-100 was added to each tube and shaking was continued for another hour. After the pre-digestion incubation, 10 μL of DpnII (was added only to each sample tube, and continued shaking until the end of the day. An additional 10 µL of DpnII was added to each digestion reaction and digestion continued overnight. The next day, another additional 10 µL of DpnII was added and the incubation continued for another 2-3 hours. 100 μL of each digestion reaction was then removed, pooled into one 1.5 mL tube. The remaining samples were heat inactivated at 65°C for 20 min at 1000 rpm in a Thermomixer, and cooled on ice for 20 additional minutes. Digested samples were ligated with 8 μL of T4 DNA ligase (HC 30U/uL) and 1X ligase buffer at 1,000 rpm overnight at 16°C in a Thermomixer. The next day, an additional 2 µL of T4 DNA ligase was spiked into each sample and incubated for another few hours. The ligated samples were then de-crosslinked overnight at 65°C with Proteinase K along with the pre-digestion and digestion controls. The following morning, both controls and ligated samples were incubated for 30 min at 37°C with RNase A, followed by phenol/chloroform/isoamyl alcohol extraction and ethanol precipitation at -20°C. The 3C libraries were centrifuged at 3000 rpm for 45 min at 4°C 14,000 rpm, to pellet the samples. DNA pellets were resuspended in 70% ethanol and again centrifuged as described above. The 3C library pellets were resuspended in 300 μL dH2O and stored at −20°C. 10 μg of isolated DNA from 3C libraries of each library was sheared in dH2O using a QSonica Q800R to an average fragment size of 350bp. After shearing, DNA was purified using AMPure XP beads. One microgram of adaptor-ligated library was used as input for the SureSelect XT capture kit using manufacturer protocol and our custom-designed 41K promoter Capture-C probe set.
Promoter Focused Capture C
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
HypothalamicProg_1frag_chicagoRes.ibed
HypothalamicProg_4frag_chicagoRes.ibed
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| Data processing |
Bases were called using Illumina bcl2fastq2 conversion v2.29
Paired-end reads from three replicates from ESCs, HPs, and HNs were pre-processed using the HICUP pipeline (v0.5.9) with the default parameters. Reads were aligned to hg19 using bowtie2. We called call significant promoter interactions using the read count from promoters included in our reference bait.
Significant interactions were called CHiCAGO (v1.1.8) with default parameters except for bin-size set to 2,500. In addition to our analysis per individual DpnII fragment (1frag), we also called interactions by binning four fragments, which improves detection of long-distance interactions. Significant interactions at 4-DpnII fragment resolution were also called using CHiCAGO with artificial baitmap and .rmap files where four DpnII fragments were concatenated. Interactions with a CHiCAGO score > 5 in at least one cell type in either 1-fragment or 4-fragment resolution were considered significant.
Genome_build: hg19
Supplementary_files_format_and_content: ibed
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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) |
| GSE152092 |
Cis-regulatory architecture of human ESC-derived hypothalamic neuron differentiation aids in variant-to-gene mapping of relevant common complex traits (Capture C) |
| 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 |
SAMN15186421 |
| SRA |
SRX8508334 |
| 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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