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Sample GSM5225708

Query DataSets for GSM5225708

Status

Public on Apr 14, 2021

Title

MCF-10A_NeuT_Control-1

Sample type

SRA

Source name

Mammary gland; breast

Organism

Homo sapiens

Characteristics

tissue: Non-tumorigenic epithelial cell line
cell line: MCF-10A
treatment: Untreated

Treatment protocol

Empty and NeuT cells were treated with 1uM of JG-98 for 12hours or left untreated which served as controls.

Growth protocol

MCF10A (human breast epithelial) cells were grown in DMEM/F-12 1:1 medium supplemented with 5% horse serum (Cat#04-0041A, BI-Biologicals), 20 ng/mL epidermal growth factor (Cat#AF-100-15,Peprotech, 1 mg), 0.5 μg/mL hydrocortisone(Sigma #H-0888), 10 μg/mL human insulin (Cat#I9278, Sigma), and 100 ng/mL cholera toxin (Sigma #C-8052), L-glutamine (Cat#03-020-1B, BI-Biologicals), L-alanyl-L-glutamine (Cat#03-022-1B, BI-Biologicals), penstrep (Cat#03-031-1B, BI-Biologicals), and were grown in a humidified incubator at 37°C and 5% CO2.

Extracted molecule

total RNA

Extraction protocol

Total RNA was isolated from cells using Qiagen RNeasy Plus Mini kit (Cat. #74134;GmbH) as per manufacturer protocol.
mRNA molecules were purified from total RNA using oligo(dT)-attached magnetic beads and fragmented into small pieces using fragmentation reagent after reaction a certain period in proper temperture. First-strand cDNA was generated using random hexamer-primed reverse transcription, followed by a second-strand cDNA synthesis. The synthesized cDNA was subjected to end-repair and then was 3’ adenylated. Adapters were ligated to the ends of these 3’ adenylated cDNA fragments. This process was to amplify the cDNA fragments with adapters from previous step. PCR products were purified with Ampure XP Beads (AGENCOURT), and dissolved in EB solution. Library was validated on the Agilent Technologies 2100 bioanalyzer. The double stranded PCR products were heat denatured and circularized by the splint oligo sequence. The single strand circle DNA (ssCir DNA) were formatted as the final library. The library was amplified with phi29 to make DNA nanoball (DNB) which had more than 300 copies of one molecular. The DNBs were load into the patterned nanoarray and single end 50 (pair end 100) bases reads were generated in the way of sequenced by synthesis.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

BGISEQ-500

Data processing

Reads were trimmed and clipped for quality control in trim_galore (v0.5.0).
Read quality was checked for each sample using FastQC (v0.11.7).
Reads were taken to Hisat2 (v2.1.0) for alignment using hg38, GRch38.97
High-quality reads were then imported into samtools (v1.9 using htslib 1.9) for conversion of SAM files to BAM file.
Gene-count summaries were generated with featureCounts (v1.6.3): A numeric matrix of raw read counts was generated, with genes in rows and samples in columns, and used for differential gene expression analysis with the Bioconductor Limma package.
Genome_build: hg38, GRch38.97
Supplementary_files_format_and_content: featurecounts_NeuTvsEmpty.txt :Combined Tab-delimited text file includes count for all samples.
Supplementary_files_format_and_content: ETEC_geneset.csv : List of differentially expressed genes in Empty_Treated (ET) compared to Empty_control (EC).
Supplementary_files_format_and_content: NTNC_geneset.csv :List of differentially expressed genes in Neut_Treated (NT) compared to Neut_control (NC).

Submission date

Apr 04, 2021

Last update date

Apr 14, 2021

Contact name

Shivani Patel

E-mail(s)

patel.shiva2593@gmail.com, shivanip@ariel.ac.il

Organization name

Ariel University

Department

Molecular Biology