GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM4105584

Query DataSets for GSM4105584

Status

Public on Apr 30, 2023

Title

M1+TAMd14_RNA-seq

Sample type

SRA

Source name

HMLE-TWIST1-ER cells, EMT-sensitive, clone 1

Organism

Homo sapiens

Characteristics

cell type: mammary epithelial cells
resistance: EMT-sensitive
treatment: 14 days Tamoxifen treatment

Treatment protocol

For TWIST1 activation, cells were treated with (Z)-4-HydroxyTamoxifen at a final concentration of 20 nM for the indicated number of days.

Growth protocol

Human mammary epithelial (HMLE) cells were cultured in Mammary Epithelial Cell Basal Medium containing 0.004 ml/ml BPE, 10 ng/ml EGF, 5 µg/ml hydrocortisone; HMLE-TWIST1-ER cells were propagated in medium containing Blasticidin S HCl at a final concentration of 10 µg/ml.

Extracted molecule

total RNA

Extraction protocol

ATAC-seq of HMLE-TWIST1-ER cells: 50000 cells were tagmented in 50 µl with 2.5 µl Tn5 according to Corces et al. 2017
RNA-seq of HMLE-TWIST1-ER cells: Total RNA was isolated using the miRNeasy Mini Kit (Qiagen).
RNA-seq of primary breast cancer cells: Total RNA was isolated using the Arcturus PicoPure RNA isolation kit.
ATAC-seq of HMLE-TWIST1-ER cells: Illumina Nextera DNA Library Preparation Kit (FC-121-1030) was used for library preparation. Libraries were selected for fragments less than 600 bp using AMPure XP beads for size selection.
RNA-seq of HMLE-TWIST1-ER cells: 1 μg of RNA was depleted for cytoplasmatic rRNAs, fragmented, and reverse transcribed with the Elute, Prime, Fragment Mix. A-tailing, adaptor ligation, and library enrichment were performed as described in the High Throughput protocol of the TruSeq RNA Sample Prep Guide (Illumina).
RNA-seq of primary breast cancer cells: Libraries were prepared from 1 μg of total RNA using a TruSeq Stranded Total RNA Kit with Ribo-Zero Human/Mouse/Rat (Illumina) according to the manufacturer’s instructions.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 4000

Description

HMLE-TWIST1-ER cells, EMT-sensitive, clone 1; 14 days Tamoxifen treatment
RNA-seq_HMLE_TWIST1_ER_all_counts.xlsx
ISF0969

Data processing

ATAC-seq of HMLE-TWSIT1-ER cells: ATAC-seq reads were aligned to the human genome (hg38) using bowtie (Langmead et al., 2009) with options “-q -n 2 --best --chunkmbs 2000 -p 32 -m1”
RNA-seq HMLE-TWIST1-ER cells: The STAR aligner (v 2.4.2a) with modified parameter settings (--twopassMode=Basic) is used for split-read alignment against the human genome assembly hg19 (GRCh37) and UCSC knownGene annotation (Dobin, A. et al. , 2013).
RNA-seq of primary breast cancer cells: RNA-sequencing reads were aligned to the human reference genome hg19 and quantified using the stranded option of STAR version 2.4.2a30.
Genome_build: ATAC-seq: hg38 RNA-seq: hg19 (GRCh37)
Supplementary_files_format_and_content: ATAC-seq of HMLE-TWIST1-ER cells: bigWig files were generated from homer tag directories using makeBigWig.pl.
RNA-seq of HMLE-TWIST1-ER cells: To quantify the number of reads mapping to annotated genes we use HTseq-count (v0.6.0) (Anders, S., Pyl, P. T. & Huber, W., 2015). FPKM (Fragments Per Kilobase of transcript per Million fragments mapped) values are calculated using custom scripts.
RNA-seq of primary breast cancer cells: The DESeq2 package was used to obtain normalized expression values as described (Raffel, S. et al., 2017).

Submission date

Oct 02, 2019

Last update date

Apr 30, 2023

Contact name

Massimo Saini

E-mail(s)

massimo.saini@unibas.ch

Phone

0762311275

Organization name

University of Basel