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Sample GSM4063265

Query DataSets for GSM4063265

Status

Public on Nov 01, 2020

Title

ST-06-38_Lu

Sample type

SRA

Source name

lung

Organism

Rattus norvegicus

Characteristics

strain: Fischer 344
Sex: male
treatment: crystalline silica & cigarette smoke

Treatment protocol

Exposures were performed in a whole-body inhalation chamber. Group 1: Six rats were exposed to air for 29 consecutive weeks. Group 2: Six rats were exposed to air for weeks 1 and 2; 15mg/m3, 6hours/day, 5days Min-U-Sil5 silica for week 3; and air for weeks 4-29. Group 3: Six rats were exposed to 40mg/m3, 3hours/day cigarette smoke on Tuesday and Friday of week 1; 80mg/m3, 3hours/day cigarette smoke on Tuesday and Friday of week 2; air for week 3; and 80mg/m3, 3hours/day cigarette smoke on Tuesday and Friday of weeks 4-29. Group 4: Six rats were exposed to 45mg/m3, 3hours/day cigarette smoke on Tuesday and Friday of week 1; 80mg/m3, 3hours/day cigarette smoke on Tuesday and Friday of week 2; 15mg/m3, 6hours/day, 5days Min-U-Sil5 silica for week 3; and 80mg/m3, 3hours/day cigarette smoke on Tuesday and Friday of weeks 4-29.

Growth protocol

This experiment was conducted in an animal facility (NIOSH, Morgantown, WV) accredited by AAALAC International following a protocol approved by the Institutional Animal Care and Use Committee (IACUC). Male Fischer (CDF®) rats (F344/DuCrl) 3 months of age were purchased from Charles River Laboratories (Wilmington, MA). Rats were housed 2 per cage in a temperature (22-25oC) and humidity (40-60%) controlled environment following a 12hr light-dark schedule with free access to Harlan Teklad Global 18% Protein Rodent Diet, 2918 irradiated rodent chow, (Harlan Laboratories, Frederick, MD, USA) and tap water. Rodents were acclimated to the facility for 10 days before treatment commenced.

Extracted molecule

total RNA

Extraction protocol

Total RNA, including miRNA and other small RNA molecules, was isolated from a piece of the lung tissue using the miRNeasy Mini Kit (Qiagen, Inc.; Valencia, CA) following the procedure provided by the manufacturer, including optional DNase treatment with RNase-Free DNase (Qiagen, Inc.; Valencia, CA).
One microgram total RNA/sample was used to create sequencing libraries using the Illumina TruSeq® Stranded Total RNA Library Prep (Illumina, Inc., San Diego, CA) following the manufacturer's protocol. Briefly, following depletion of ribosomal RNA (rRNA), the RNA samples were purified, fragmented (68°C for 5 minutes), and primed for cDNA synthesis. The denatured and cleaved RNA fragments were purified using a bead cleanup and reverse transcribed into first strand cDNA using reverse transcriptase and random primers. While synthesizing the double stranded cDNA, dUPT was incorporated in place of dTTP followed by the addition of a single ‘A’ nucleotide to the 3 prime ends to facilitate proper adapter ligation to each sample. Indexing adapters provided in the library preparation kit were ligated to the ends of the ds cDNA. After adapter ligation, the samples were PCR amplified (12 cycles) to enrich the DNA fragments containing the adapter molecules and to enhance the amount of DNA in the library using a Veriti™ 96 Well Thermal Cycler (Applied Biosystems; Foster City, CA).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Description

total RNA including small RNAs
ST06Lu_Counts.txt
ST_06_BL_038_ST

Data processing

Basecalls were performed using bcl2fastq/2.19.
Sequence reads were trimmed for adaptor sequence low-quality using Trimmomatic/0.35 with the following parameters ILLUMINACLIP:adapters/TruSeq2-PE.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:85
Sequences were mapped to Rnor6.0 whole genome using Hisat2/2.1.0 with the following parameters: -q --downstream-transcriptome-assembly --dta-cufflinks --fr --no-mixed --time --met-file
Aligned sequences were identified, sorted, and counted using Samtools/1.8 and HTSeq/0.11.2
Genome_build: Rnor6.0
Supplementary_files_format_and_content: tab-delimted text file including raw counts in a matrix containing all samples

Submission date

Sep 05, 2019

Last update date

Nov 01, 2020

Contact name

Christina M Umbright

Organization name

DHHS/PHS/CDC/NIOSH

Department

HELD

Lab

L-4324