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Sample GSM5579318

Query DataSets for GSM5579318

Status

Public on Jun 15, 2022

Title

Animal_Fulv10

Sample type

SRA

Source name

Liver/ liver tumor (MCF7-ESR1Y537S)

Organisms

Homo sapiens; Mus musculus

Characteristics

Sex: female
treatment: Fulvestrant
tissue: Liver/ liver tumor
strain: NOD SCID gamma (NSG)/ ATCC HTB-22 (RRID:CVCL_0031)

Biomaterial provider

Jackson Laboratory

Treatment protocol

In vivo study: four-week-old, ovariectomized, NOD SCID gamma (NSG) immunodeficient female mice were obtained from The Jackson Laboratory (Bar Harbor, ME).After one week of acclimatization to the housing facility, we injected 1×106 MCF7-ESR1Y537S cells resuspended in 1% PBS via tail vein and randomized animals to indicated treatment groups.We randomized N=8 mice to one of the two treatments Vehicle (Veh) or Fulv in each diet group. Fulv (Sigma) was dissolved in 10% DMSO and 90% corn oil and administrated via intramuscular injection (100 mg/kg) twice a week (Monday, Friday) for four weeks. In vitro study, IN SITE Metastasis Kit (Xylyx Bio, Inc., NY) containing TissueSpec Bone (MTSBN101), Liver (MTSLV101), and Lung (MTSLG101) ECM Hydrogels, were used to model tumor microenvironments according to the manufacturer’s protocol. Briefly, 2×103 cells were encapsulated in corresponding extracellular matrix (ECM) hydrogels by mixing them with tissue culture matrix. A mixture volume of 100 uL/well was placed in 96-well plates in triplicate. Plates were incubated at 37°C in a humidified environment with 5% CO2 for at least 45 minutes to achieve gelation. Cells were treated with media containing Veh or 1μM Fulv every Monday and Friday for three weeks.

Growth protocol

MCF7 (ATCC HTB-22) (RRID:CVCL_0031) and T47D (ATCC HTB-133) (RRID:CVCL_0553) parental cells were cultured in RPMI-1640 medium with NEAA salts (Sigma, St Louis, MO, USA), 5% fetal bovine serum (FBS) (HyClone, Logan, UT), 100 μg/mL penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA) and 50 mg/mL Gentamicin (Gibco, Gaithersburg, MD). MCF7/ESR1D537S and - ESR1Y537S cells (RRID:CVCL_0031-citation needed) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) with NEAA salts, 10% FBS, 100 μg/mL penicillin/streptomycin, and 50 mg/mL gentamicin. T47D ESR1D537S and T47D ESR1Y537S cells (RRID: CVCL_0553-citation needed) were cultured in Modified Eagle Medium (MEM) with NEAA salts, 10% FBS, 100 μg/mL penicillin/streptomycin, and 50 mg/mL gentamicin. Cell line authentication was performed by checking activity and expression of ERα, proliferative responses to ER agonists and antagonists for all cell lines, and sequencing of MCF7/ESR1Y537S and MCF7/ESR1D537G as described.

Extracted molecule

total RNA

Extraction protocol

RNA extraction: For gene expression analysis, total RNA was extracted from at least 3 biological replicates for each ligand treatment using Trizol reagent and further cleaned using the RNAeasy kit from QIAGEN. ChIP-seq analysis was performed as described previously using MCF7-ESR1Y537S cells. ChIP: ERα–DNA or IgG–DNA complexes were immunoprecipitated using ERα- specific F10 and HC20 antibodies (Santa Cruz Biotech, 3:100 dilution).
Once the sample quality and replicate reproducibility was verified at least 2 samples from each group were subjected to sequencing. RNA at a concentration of 100 ng/µL in nuclease-free water was used for library construction.The RNAseq libraries were prepared with Illumina's 'TruSeq Stranded RNAseq Sample Prep kit. The libraries were quantitated by qPCR and sequenced on one lane for 101 cycles from each end of the fragments on a HiSeq2000 using a TruSeq SBS sequencing kit version 3. Fastq files were generated with the software Casava 1.8.2 (Illumina). Briefly, the poly-A containing mRNA was purified from total RNA, RNA was fragmented, double-stranded cDNA was generated from fragmented RNA, and adapters were ligated to the ends. ChIP DNA was obtained from three pooled biological replicates. Libraries were prepared according to Illumina Solexa ChIP-Seq sample processing (San Diego, CA), and single-read sequencing was performed using the Illumina HiSeq 2000. Sequences generated were mapped uniquely onto the human genome (hg18) by Bowtie2 (RRID:SCR_016368). The MACS (model-based analysis of ChIP-seq) algorithm was used to identify enriched peak regions (default settings) with a P value cutoff of 6.0e−7 and FDR of 0.01, as we have described.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

RNAseq_Animal_Fulv
Aligned_human_rnaseq_featurecounts.txt

Data processing

Reads from the HiSeq 2000 were processed and analyzed through a series of steps. Base calling and de-multiplexing of samples within each lane were done with Casava 1.8.2.
For RNAseq, STAR (version 2.7.6a) was employed to map RNA-Seq reads to either version mm39 of the Mus musculus reference genome, GRCm39, from NCBI Genome Reference Consortium Mouse Build 39 or version GRCh38.p13 of the human reference genome from NCBI Genome Reference Consortium Human Build 38 patch release 13. For ChIPseq, Bowtie2 was employed to obtain BAM files.
For RNAseq, gene expression values (raw read counts) from BAM files were calculated using FeatureCounts function from the Subread module (version 2.0.0). For ChIPseq, gene expression values (raw read counts) from BAM files were calculated using BigWig and outputed as BW files.
DESeq normalization algorithm with DESeq2 module in R using default values was used. Differentially expressed genes were then determined by fold-change and p-value for each gene for each treatment relative to the vehicle control.
Genome_build: mm39 for mouse, GRCh38.p13 for human

Submission date

Sep 15, 2021

Last update date

Jun 15, 2022

Contact name

Zeynep Madak Erdogan

Organization name

UIUC

Department

FSHN

Lab

Zeynep Madak Erdogan