|
| Status |
Public on Jan 01, 2021 |
| Title |
hDF_F64Br_Control_12h_rep2 |
| Sample type |
RNA |
| |
|
| Source name |
hDF_F64Br_Control_12h
|
| Organism |
Homo sapiens |
| Characteristics |
donor id: F64Br
Sex: Female
age: 64y
tissue: Breast
cell type: enzyme-derived, human dermal fibroblasts (hDF)
treatment: Control
treatment time: 12h
|
| Treatment protocol |
hDF were seeded into 6-well microplates and cultured in complete DMEM for four days at 37°C in 5% CO2. hDF were equilibrated in low (1%) serum DMEM for 24h and treated with or without TGF-β1 (10 ng/ml; R&D) in low (1%) serum DMEM for a further 12h or 24h.
|
| Growth protocol |
Human dermal fibroblasts (hDF) were cultured in phenol red-free, low glucose DMEM (Sigma-Aldrich) supplemented with 10% foetal bovine serum; 450 µg/ml L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin (Sigma-Aldrich). Referred to as complete DMEM.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using the RNeasy mini kit (Qiagen) according to the manufacturer’s instructions. RNA integrity was measured using the Bioanalyser 2100 (Agilent) and RNA 6000 Nano kit (Agilent) according to the manufacturer’s instructions.
|
| Label |
Cy3
|
| Label protocol |
One colour Quick-Amp labelling kit (Agilent) was used to derive cyanine 3 (Cy3)-labelled cRNA from the extracted RNA. A one colour RNA spike-in kit (Agilent) was used to monitor sample amplification and labelling efficiency. For each sample, 300 ng total RNA was used; universal human reference RNA (Agilent) was used as a reference. The concentration of labelled cRNA was measured using a NanoDrop 2000 spectrophotometer (Thermo Scientific).
|
| |
|
| Hybridization protocol |
Sample preparations were mixed, collected and incubated at 60°C for 30 min to fragment the cRNA using the gene expression hybridisation kit (Agilent): Cy3-labelled cRNA (600 ng) with nuclease-free water, 2X blocking agent, 1X fragmentation buffer. The fragmentation reaction was stopped by adding 1X hybridisation buffer Hi-RPM. Each SurePrint microarray (Agilent) was incubated in a rotisserie oven at 65°C for 17h to allow hybridisation. Following hybridisation, microarrays were added to gene expression wash buffer 1 (Agilent), incubated at room temperature for 1 min. Microarrays were washed in wash buffer 2 (Agilent) at 37°C for 1 min. Arrays were incubated in acetonitrile (Sigma-Aldrich) at room temperature for 10 sec, dried and covered with ozone protection covers (Agilent).
|
| Scan protocol |
Arrays were imaged using the Agilent G2565BA microarray scanner system. All three experiments were run using the SurePrint G3 Human GE 8x60K V2 kit (Agilent).
|
| Description |
F64Br_Control12(normalized)
|
| Data processing |
Scanned images were analysed with G2567AA feature extraction software (v.9.5.1; Agilent) using default parameters (protocols GE1-v5_91_0806; grid 039494_D_F_20150612). Gene expression values were normalised using Agilent GeneSpring GX software (v.14.8).
|
| |
|
| Submission date |
Nov 25, 2019 |
| Last update date |
Jan 02, 2021 |
| Contact name |
Oliver J Culley |
| E-mail(s) |
oliver.culley1@gmail.com
|
| Phone |
+447500 939574
|
| Organization name |
King’s College London
|
| Department |
Centre for Stem Cells and Regenerative Medicine
|
| Lab |
Fiona M Watt
|
| Street address |
Floor 28, Tower Wing, Guy’s Hospital, Great Maze Pond
|
| City |
London |
| ZIP/Postal code |
SE1 9RT |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL17077 |
| Series (1) |
| GSE140962 |
Correlating functional heterogeneity of human dermal fibroblasts with differences in gene expression |
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