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Status |
Public on Jun 15, 2018 |
Title |
N182-C3 |
Sample type |
SRA |
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Source name |
breast
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Organism |
Homo sapiens |
Characteristics |
cell type: epithelial treatment: control time: 0 hr
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Treatment protocol |
3D cultures from 3 specimens were exposed to 10nM estradiol or vehicle alone for 6 or 24 hours.
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Growth protocol |
The wells of a 24-well cell culture plate were uniformly coated with 50µl lrECM (BME, Matrigel Basement Membrane Matrix Growth Factor Reduced, Corning) /well and allowed to polymerize at 37°C for 20 min. Organoid aliquots were thawed, diluted in baseline medium: Advanced DMEM/F12 (ThermoFisher) containing 30mM HEPES (Sigma), 1x GlutaMAX (ThermoFisher), 140 nM hydrocortisone (Sigma), 10 ng/ml EGF (Sigma), 1% PenStrep (ThermoFisher), and centrifuged twice before resuspension in 600 µl lrECM. 50µl portions were plated at the centers of the pre-coated wells. Following 1h polymerization, 500µl of pre-warmed culture medium was added to each well and organoids were incubated for 2 days at 37°C, 5% CO2 in a humidified incubator. Organoids were retrieved from lrECM by removing medium and adding 500µl of Cell Harvesting Buffer/Dispase (Corning) to each well and incubating for 1h at 37°C. Organoids were then collected and mixed with an equal volume of 0.5 mM EDTA in phosphate buffered saline (PBS), and incubated for an additional 15 minutes at room temperature before centrifugation. Organoids were then dissociated to single cells using 1ml of TryPLE (ThermoFisher) for 10 min at 37°C, while shaking. Following dissociation, the suspension was filtered through 35µm nylon mesh to obtain single cells. The cells were then washed in baseline medium, resuspended in lrECM and replated in pre-coated 24-well plates at a density of 25,000 cells/well. Following lrECM polymerization for 1 h, 500 µl medium was added per well, with indicated additional factors including EGF (50 ng/ml, Sigma), Noggin (100 ng/ml, PeproTech), R-Spondin-1 (500 ng/ml, PeproTech), Neuregulin1 (100 ng/ml, PeproTech), Jagged-1 (1 µM, AnaSpec), A 83-01 (500 nM, Tocris), SB 431542 (10 µM, Sigma), RepSox (25 µM, Sigma). ROCK inhibitor Y-27632 (10 µM, Tocris) was added to all the cultures during dissociation and for the first 2-3 days of culture. Cells were incubated for 8 to 14 days (until the mean diameter of colonies was > 100 µm) and medium was changed every other day.
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Extracted molecule |
total RNA |
Extraction protocol |
RNA was isolated directly from intact cultures using RNA STAT-60 (Amsbio) following the manufacturer’s instructions, and quantitated using a Nanodrop ND-1000 spectrophotometer (Thermo Fisher). RNA-seq libraries were prepared using the NEBNext Ultra II RNA Library Prep Kit for Illumina (New England Biolabs) from 1 µg of total RNA input per sample, following the manufacturer’s instructions.
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina HiSeq 3000 |
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Data processing |
Basecalls performed using CASAVA version 1.9 Reads were checked for per base/per tile sequence quality, per sequence GC content, adapter content etc. using FASTQC software HISAT2/htseq-count pipeline with default parameters for PE reads alignments was applied assuming that a gene is an union of its exons Raw count table was pre-filtered removing rows with all zeros through all samples. R environment and package DESeq2(Bioconductor) were used to quantify candidate genes with cutoffs for non-specific filtering and p-values = 0.1 Normalizing filtered raw counts using variance-stabilizing transformation Genome_build: hg38 Supplementary_files_format_and_content: csv-files(comma-delimited) were generated with R
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Submission date |
May 30, 2018 |
Last update date |
Jun 15, 2018 |
Contact name |
Paul Yaswen |
E-mail(s) |
P_Yaswen@lbl.gov
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Phone |
5107346880
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Organization name |
LBNL
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Street address |
One Cyclotron Road, Building 977
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City |
Berkeley |
State/province |
California |
ZIP/Postal code |
94720 |
Country |
USA |
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Platform ID |
GPL21290 |
Series (1) |
GSE115112 |
Estrogen responsive transcriptome of estrogen receptor positive normal human breast cells in 3D cultures |
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Relations |
BioSample |
SAMN09283771 |
SRA |
SRX4142753 |
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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