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Sample GSM3165453

Query DataSets for GSM3165453

Status

Public on Jun 15, 2018

Title

N182-C2

Sample type

SRA

Source name

breast

Organism

Homo sapiens

Characteristics

cell type: epithelial
treatment: control
time: 0 hr

Treatment protocol

3D cultures from 3 specimens were exposed to 10nM estradiol or vehicle alone for 6 or 24 hours.

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.

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.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 3000

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

Submission date

May 30, 2018

Last update date

Jun 15, 2018

Contact name

Paul Yaswen

E-mail(s)

P_Yaswen@lbl.gov

Phone

5107346880

Organization name

LBNL