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Sample GSM7899082

Query DataSets for GSM7899082

Status

Public on Nov 16, 2023

Title

Hypothalamus, acrolein (Hy10_S3)

Sample type

SRA

Source name

Hypothalamus

Organism

Rattus norvegicus

Characteristics

tissue: Hypothalamus
strain: Wistar-Kyoto
treatment: acrolein

Extracted molecule

total RNA

Extraction protocol

Uniform portions of frozen tissue were sectioned and processed with RNeasy non-fibrous, fibrous, or lipid tissue mini kits (Qiagen, Valencia, CA) following manufacturer directions (kit dependent upon tissue type). RNA quantity and purity (260/230 and 260/280 ratios) were spectrophotometrically quantified using a Nanodrop 1000 (ThermoFisher Scientific Inc., Waltham, MA). RNA integrity was assessed by the RNA 6000 LabChip® kit and a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA) using a RIN cutoff of 7. Samples processing for mRNA sequencing was performed on the Apollo324 automated system (Takara Bio Inc., Kusatsu, Japan) for library prep with PrepX mRNA 48 protocol v19, using the PrepXTM RNA-seq for Illumina Library Kit (Takara), SuperScript III reverse transcriptase (ThermoFisher), and AMPure XP Beads (Agilent). PCR amplification with 48 index primers was run for 16 cycles and resulting PCR product quality was analyzed again via Qubit (ThermoFisher) and bioanalyzer.
An RNA-seq library was prepared with Wafergen’s PrepX mRNA 48 protocol and dsDNA products were prepared from cDNA. Each library was sequenced according to Illumina NextSeq 500, with a final concentration of 2.2 pM + 2% PhiX and run for 75 cycles (Illumina Inc., San Diego, CA).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Description

HT_normcountsXL.csv

Data processing

Sequenced mRNA reads were mapped to the rat genome (rn7) using ensemble release 105 (Partek Flow suite, R version 4.1.3), resulting in 24,091 identified genes. Genes with fewer than 1 read in 10 were removed, leaving 20,580 genes. An average of 15.5 million reads were mapped per sample with a standard deviation of 5.5 million. PCA was performed to identify any samples that did not cluster within tissue and should be removed prior to analysis. After removal of those tissues, remaining samples had 15.8 million reads per sample with a standard deviation of 5.3 million, with an average read per gene of 766. DEGs were assessed in DESeq2 package in R (v1.34.0) [81] and normalized reads were assessed for air vs. acrolein effects.
Assembly: rat genome (rn7)
Supplementary files format and content: Processed data is provided as normalized transcript counts as a .csv file for each tissue.

Submission date

Nov 14, 2023

Last update date

Nov 16, 2023

Contact name

Devin Issac Alewel

E-mail(s)

[email protected]

Phone

9197240340

Organization name

U.S. EPA