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| Status |
Public on Jan 15, 2021 |
| Title |
MSC dervived Adipocyte donor 8002 [Capture C] |
| Sample type |
SRA |
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| Source name |
Adipocytes dervived from MSCs from donor 8002
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| Organism |
Homo sapiens |
| Characteristics |
subject status/id: healthy donor 8002
cell type: Adipocytes dervived from mesenchymal stem cells (MSCs)
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| Treatment protocol |
Adipogenic differentiation of the hMSCs was carried out 2 days after seeding using standard procedure for human cells. Briefly, cell monolayers were washed twice with phosphate buffered saline and supplemented with alpha-MEM consisting of 8.25% FBS, 0.5 µM water-soluble Dexamethasone, 0.5 µM isobutylmethylxanthine, and 50 µM Indomethacin. Alpha-MEM consisting of 8.25% FBS with or without supplementation were exchanged every 2–3 days until harvested.
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| Growth protocol |
Primary bone-marrow derived human MSCs isolated from healthy donors (age range: 22 –29 years) were characterized for cell surface expression (CD166+CD90+CD105+/CD36−CD34−CD10−CD11b−CD45−) at the Institute of Regenerative Medicine, Texas A&M University. Expansion and maintenance of the cells were carried out using alpha-MEM supplemented with 16.5% FBS in standard culture conditions by plating cells at a density of 3000 cells/cm^2.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were fixed after 2 weeks of adipogenic induction. Cells were collected and single-cell suspension were made with aliquots of 10^7 cells in 10 ml media (e.g. RPMI + 10%FCS). 540 µl 37% formaldehyde was added and incubated for 10 min at RT. The reaction was quenched by adding 1.5 ml 1 M cold glycine (4 °C) for a total volume of 12 ml. Fixed cells were centrifuged at 1000 rpm for 5 min at 4 °C and supernatant removed. The cell pellets were washed in 10 ml cold PBS (4 °C) followed by centrifugation. Supernatant was removed and cell pellets were resuspended in 5 ml of cold lysis buffer (10 mM Tris pH8, 10 mM NaCl, 0.2% NP-40 (Igepal) supplemented with protease inhibitor cocktails). Resuspended cells were incubated for 20 min on ice, centrifuged as above, and the lysis buffer removed. Finally, cell pellets were resuspended in 1 ml fresh lysis buffer, transferred to 1.5 ml Eppendorf tubes and snap frozen (ethanol/dry ice or liquid nitrogen). Cells were stored at −80 °C until they were thawed for 3C library generation.
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.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
library strategy: Capture C
Bases were called using Illumina bcl2fastq2 conversion v2.29
Paired-end reads from three replicates from MSC-dervived Adipocytes 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 |
Jan 15, 2021 |
| Last update date |
Jan 17, 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) |
| GSE164911 |
Biological constraints on GWAS SNPs at suggestive significance thresholds reveal true BMI loci [hMSC_adipocytes_captureC] |
| GSE164912 |
Biological constraints on GWAS SNPs at suggestive significance thresholds reveal true BMI loci. |
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| Relations |
| BioSample |
SAMN17330165 |
| SRA |
SRX9857070 |
| 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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