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Sample GSM3484484

Query DataSets for GSM3484484

Status

Public on Jul 17, 2019

Title

MCF7_WM7d_CD44H

Sample type

SRA

Source name

ERα positive breast cancer cells

Organism

Homo sapiens

Characteristics

cell line: MCF7 CD44high facs-sorted
treatment: E2 starvation for 7 days

Treatment protocol

MCF7 cells were treated with E2 depleted medium at 2, 4, and 7 days and sorted into CD44high and CD44low subpopulation.

Growth protocol

MCF7 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal calf serum (FCS). Long-term oestrogen-deprived cells (LTED) were derived from MCF7 after one year oestrogen deprivation as previously described and were maintained in phenol-red free DMEM containing 10% charcoal stripped fetal calf serum (SFCS) (PMID: 26610607). Both media were supplemented with 2 mM L-glutamine, 100 units/mL penicillin and streptomycin. 10−8 M estradiol (E2758 Sigma) was added routinely to MCF7.

Extracted molecule

polyA RNA

Extraction protocol

Single cells were prepared from a full population of MCF7 or LTED, or from sorting MCF7 CD44-GFP reporter cells by the level of GFP expression at different time points of E2 deprivation. After centrifugation, single cells were washed with PBS and were re-suspended with a buffer (Ca++/Mg++ free PBS + 0.04% BSA) at 1,000 cells/µl. Captures were performed using the 10x Genomics Chromium platform, according to manufacturer guidelines and in order to obtain 5,000 cells per capture.
Libraries were generated using the 10x Genomics Chromium platform.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 4000

Description

expression_matrix.rawCounts.txt.gz
expression_matrix.normalizedCounts.txt.gz

Data processing

cellRanger (v2.1.1) was run on the raw data using GRCh38 annotation (v1.2.0). Output from cellRanger was loaded into R using the function load_cellranger_matrix_h5 from package cellranger (v1.1.0; genome = ”GRCh38”). Datasets were merged according to gene names.
All cells sampled were retained except for flow-sorted CD44high and CD44low either in +E2 media or starved for two days, for which the top 5,000 cells in terms of UMIs per cell were considered.
A filter on cells showing at least 1,500 detected genes per cell and at least 5,000 UMIs per cell was then applied. After that, reads mapping on mitochondrial genes were excluded. Before normalization, data were imported in Seurat (v2.3.4; PMID: 29608179) and scaled (NormalizeData function using normalization.method = "LogNormalize", scale.factor = 10000, followed by the ScaleData function).
A filtering step was then performed based on the cumulative level of expression (the sum of the Seurat-scaled values) of three housekeeping genes (GAPDH, RPL26 and RPL36, http://doi.org/10.1101/229815). Cells showing housekeeping gene expression in the bottom 1% were then excluded from further analyses. At last, genes expressed in less than 20 cells were excluded. Across-cells normalization was performed using the R package Scran (v1.6.9) (PMID: 27909575).
Raw counts were imported into a SCE object using the newSCESet function; size factors were calculated using computeSumFactors (sizes = seq(20, 250, 10)), on data pre-clustered through quickCluster.
Genome_build: GRCh38
Supplementary_files_format_and_content: expression_matrix.rawCounts.txt: expression matrix (raw counts), flat text file with tab delimiters; expression_matrix.normalizedCounts.txt: expression matrix (normalized counts), flat text file with tab delimiters.

Submission date

Nov 20, 2018

Last update date

Jul 17, 2019

Contact name

Iros Barozzi

E-mail(s)

iros.barozzi@meduniwien.ac.at

Organization name

Medical University Vienna

Street address

Borschkegasse 8a