|
| Status |
Public on Aug 12, 2020 |
| Title |
Fibroblasts AD-HIES1_TNF |
| Sample type |
SRA |
| |
|
| Source name |
skin biopsy
|
| Organism |
Homo sapiens |
| Characteristics |
isolated cells: skin fibroblasts
treatment: TNFa 50ng/ml for 8h
genotype/variation: AD-HIES STAT3 mutation
|
| Treatment protocol |
The fibroblasts were routinely grown in DMEM medium supplemented with 10% fetal bovine serum and seeded for experiments on six well plates at a density of 250,000 cells per well and switched to serum free medium after they reached confluence. After 24h in serum free medium, media was replaced with fresh serum free media and treated of not with TNFa 50ng/ml for 8h
|
| Growth protocol |
3 Control and 3 AD-HIES patient-derived fibroblasts lines were generated from 3-4-mm skin biopsies following informed consent under protocols approved by NHLBI IRB. The skin biopsy was cut into 1mm pieces and digested for 1h at 37C in 10ml of 0.1% Collagenase Type II (No.17101-015, Thermo Fisher Scientific) / 0.25 U/ml Dispase (No. 17105-0411, Thermo Fisher Scientific)/PBS solution. The skin pieces were then transferred to 2 wells of a 6 well culture plate, covered with cover slips to facilitate attachment, and cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 20% fetal bovine serum and antibiotics in 20% O2, 5% CO2 incubator. Fibroblasts outgrown from the explants were subcultured after 3-4 weeks when they occupied most of the well’s surface. The fibroblasts then were cultured in DMEM medium supplemented with 10% fetal bovine serum (No. S10250, Atlanta biological, Flowery Branch, GA) and antibiotics.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using TRIzol Reagent (No. 15596026, ThermoFisher Scientific).
One mg of total RNA was used as input for library preparation using the TruSeqRNA Sample Preparation Kit (Illumina) according to manufacturer’s instructions.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
Fibroblasts_RNA-seq.txt
|
| Data processing |
RNA-seq data quality was assessed using FastQC
Reads obtained for each sample were aligned to the hg19 human reference genome using Tophat2
Genome_build: hg19
Supplementary_files_format_and_content: tab-delimited text file include total counts values for all samples
|
| |
|
| Submission date |
Oct 21, 2019 |
| Last update date |
Aug 12, 2020 |
| Contact name |
Manfred Boehm |
| Organization name |
NHLBI
|
| Street address |
9000 Rockbille Pike
|
| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
| |
|
| Platform ID |
GPL11154 |
| Series (2) |
| GSE139202 |
Analysis of gene expression in skin fibroblasts derived from patients with Autosomal-dominant hyper-IgE syndrome (AD-HIES) |
| GSE139365 |
Gene expression analysis in primary cells derived from patients with Autosomal-dominant hyper-IgE syndrome (AD-HIES) |
|
| Relations |
| BioSample |
SAMN13071260 |
