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Sample GSM1561390

Query DataSets for GSM1561390

Status

Public on Mar 02, 2015

Title

LP-C1

Sample type

SRA

Source name

Immortalized mesothelial cell line LP9, untreated

Organism

Homo sapiens

Characteristics

cell line: LP9
cell type: immortalized mesothelial cells
cell source: peritoneal cavity
treatment: no exposure for 8 hrs

Treatment protocol

All cells were incubated at 37°C in 5% CO2 and grown to 80-90% confluence before addition of crocidolite asbestos fibers prepared and added (5 μg/cm2 or 75 x 10^6 μm2/cm2 for 8 h) to cell culture medium. After sterilization under ultraviolet light overnight to avoid endotoxin and microbial contamination, fibers were suspended in HBSS at 1 mg/ml, sonicated for 15 minutes in a water bath sonicator, and triturated five times through a 22-gauge needle. This suspension was added to cells in medium.

Growth protocol

LP9 cells were grown in 50:50 DMEM/F-12 medium containing 10% FBS, and supplemented with penicillin (50 units/ml), streptomycin (100 mg/ml), hydrocortisone (100 mg/ml), insulin (2.5 mg/ml), transferrin (2.5 mg/ml), and selenium (2.5 mg/ml); all other cells were grown in 50:50 M199:MCDB106 medium supplemented with 15% FBS, 10 ng/mL EGF, 0.4 µg/mL hydrocortisone, 50 units/mL penicillin and 100 µg/mL streptomycin.

Extracted molecule

total RNA

Extraction protocol

1 μg of total RNA was subjected to library synthesis using the TruSeq V2 RNA-Seq kit.
1 μg of total RNA was enriched for the Poly-A mRNA and reverse transcribed to double-stranded cDNA. The cDNA was end repaired, adenylated, and ligated with Illumina indexes prior to 12 cycles of PCR. Sequencing was performed using 12pM hybridization to a 2x100 paired end flow cell.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 1000

Description

2x100 paired-end sequence reads

Data processing

Illumina HiSeq 1000 reads were trimmed and clipped for quality control in Trimmomatic v0.27.
Read quality was checked for each sample using FastQC v0.10.1.
High-quality reads were then imported into TopHat v2.0.8 for alignment into BAM files.
BAM files were imported into the RNA-Seq pipeline of Partek Genomics Suite, version 6.6 (Copyright 2009, Partek Inc., St. Louis, MO, USA), and RPKM counts were calculated (RefSeq release 2013-5-10). Exclusions included a minimum RPKM value of 1.0 in at least two of the 16 samples (potentially duplicates of one sample group). RPKM values were log2-transformed with an offset value of 1.0.
Genome_build: Alignment was initially to the transcriptome (hg19, GRCh37.72), and then to the genome (hg19, GRCh37).
Supplementary_files_format_and_content: ProcessedDataMatrix_Final.txt: Tab-delimited text file includes RPKMs (log2-transformed with an offset value of 1.0) for each sample.

Submission date

Dec 08, 2014

Last update date

May 15, 2019

Contact name

Julie Dragon

E-mail(s)

julie.dragon@uvm.edu

Organization name

UNIVERSITY OF VERMONT & ST AGRIC COLLEGE

Department

MMG