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Sample GSM4520610

Query DataSets for GSM4520610

Status

Public on Aug 11, 2020

Title

S22_LUHMES_diff_DopaNeuron_Methyl_mercury_1

Sample type

SRA

Source name

dopa-neurons exposed to 0.5uM MHG

Organism

Homo sapiens

Characteristics

cell line: Lund human mesencephalic (LUHMES) cell line
cell type: Dopaminergic Neurons Differentiated from LUHMES Cells
compound treatment: Methyl_mercury
compound name: Methylmercury Chloride
publication comp. abbr.: MHG
toxicant dose: 0.5uM
teatment class: Toxicant
cas#: 115-09-3

Treatment protocol

For gene expression assays, 3 x 10e5 cells were cultured per well in 12 well plates with 2 mL medium to enable RNA extraction. Toxicant stock solutions in DMSO were diluted with culture medium to make 10X working solutions for each concentration and added to the cells at 10% of the volume in each well. Control cells were treated with 1% DMSO to match treatments. Cells were treated with DMSO or a toxicant for 6h prior to commencing RNASeq sample preparation.

Growth protocol

LUHMES cells were grown in culture vessels coated with 50mg/ml each poly-L-ornithine and human fibronectin in Advanced DMEM/F12 medium containing 2 mM L-glutamine, 1X N2-supplement and 40 ng/ml bFGF. LUHMES differentiation was induced by 1mg/ml tetracycline and 1 mM cAMP contained in advanced DMEM/F12 medium with 2 mM L-glutamine, 1X N2-supplement, and 2ng/ml GDNF (Schildknecht et al. 2009).

Extracted molecule

polyA RNA

Extraction protocol

Initial extraction and polyA enrichment were performed in the NCATS laboratories. Total RNA samples were prepared from cultured cells using Qiaquick kit (Qiagen, Germantown, MD) and selected for polyA+ transcripts using the Qiagen Oligotex midi kit. The resulting samples were then depleted of ribosomal sequences using two rounds of RiboZero Gold™, Illumina Inc. TruSeq Protocol by the NHLBI-NIH sequencing core (NHLBI-NIH DNA Sequencing and Genomics Core, Bethesda, MD, https://www.nhlbi.nih.gov/science/dna-sequencing-and-genomics-core).
RNA libraries were prepared for sequencing using standard Illumina protocols (TruSeq)
RNA-Seq library production (TruSeq) for Hiseq 3000

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 3000

Data processing

Fastq-gz files were delivered from the NHLBI sequencing core. All processing was performed at the NIH HPC resource. Paired end sequence reads files, three file pairs from three lanes per sample, were aligned to the Ensembl GRCh38 Human Genome sequence using STAR aligner.
Alignment BAM files for each biological sample were merged to a single BAM file for each biological sample.
Merged BAM files, one per sample, were analyzed by htseq-count script (HTSeq) to produce raw count files.
Individual count files for all samples were combined to produce the raw, unnormalized count matrix (See Raw Matrix File).
DESeq was used to normalize read counts to produce a normalized count matrix (see Normalized Matrix File).
Differential expression tests and further analysis are described in the associated publication.
Genome_build: GRCh38
Supplementary_files_format_and_content: Raw unnormalized matrix file. A tab delimited text file containing Ensembl Gene ids and Gene Symbols, one row per gene, 24 columns of data, one per sample. Values are un-normalized read counts per gene in each sample.
Supplementary_files_format_and_content: Normalized matrix file. A tab delimited text file containing Ensembl Gene ids and Gene Symbols, one row per gene, 24 columns of data, one per sample. Values are DESeq normalized read counts per gene in each sample.

Submission date

May 06, 2020

Last update date

Aug 11, 2020

Contact name

David Lee Gerhold

E-mail(s)

[email protected]

Phone

301-827-5346

Organization name

NCATS/NIH