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Sample GSM7719531

Query DataSets for GSM7719531

Status

Public on Jul 18, 2024

Title

Right kidney, Unilateral nephrectomised, 48h, rep3, RNAseq

Sample type

SRA

Source name

cross sectional sample

Organism

Mus musculus

Characteristics

tissue: Right kidney
tissue: cross sectional sample
strain: C57BL/6
treatment: Unilateral nephrectomy
time post_surgery: 48h
treatment: Unilateral nephrectomy
gender: Male
mouse id: 7
batch: 2
age: 8-12 weeks
library kit: Illumina TruSeq Stranded mRNA library preparation kit
bp size: 150

Extracted molecule

polyA RNA

Extraction protocol

Kidney cross-section were freshly harvested at various timepoints after sham surgery or nephrectomy and tissue samples were flash frozen in liquid nitrogen. Samples were stored at -80°C until extraction. Prior to extraction the frozen kidney samples were immersed overnight at -20°C in RNAlater -ICE (Thermo Fisher AM7030) as per the manufacturer’s instructions to transition the tissue to a state that was more amenable to pestle homogenisation. Total RNA was isolated from the kidney samples using the mirVana isolation kit (AM1561) as per the manufacturer’s instructions except that Trizol reagent was used instead of phenol. RNA was eluted in 35µl of elution buffer. The LabChip GX Nucleic Acid Analyser (Perkin Elmer) and the Qubit RNA HS (Thermo Fisher Q32852) were used according to the manufacturer’s instructions to quantify the RNA.
Library preparation was performed using the Illumina TruSeq Stranded mRNA library preparation kit. Briefly, poly-A containing mRNA molecules were purified, fragmented, and converted to strand specific cDNA. This was followed by second strand cDNA synthesis, adapter ligation and PCR amplification. Resulting libraries were quality controlled using the Qubit 2.0 (Thermo Fisher Scientific) for quantitation and the LabChip GX Touch 24 (Perkin Elmer) for quality assessment. Individual libraries were combined into a final equimolar pool and then sequenced using 150bp paired end runs on the NovaSeq system (Illumina).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Description

RNASeq-3
RNASeq-3

Data processing

RNA-seq raw data in FASTQ format from individual libraries were uploaded to Deep Thought High Performance Compute Server, hosted by Flinders University for processing. FastQC (v0.11.8) was used for checking the quality of the FASTQ file. After inspection of data quality, low quality bases and Illumina Truseq adapters were then removed using AdapterRemoval version 2.3.2 with parameters --trimns --minquality 30 --minlength 50. Quantification of transcript expression in RNA-seq data was conducted using Salmon version 1.5.0 using parameters -libType A --seqBias --numBootstraps 50 –validate Mappings. First, a decoy-aware transcriptome index was built using transcript sequences and primary assembly genome sequences from GENCODE Mouse Release M27. RNA-seq reads were then pseudoaligned to the transcriptome index and output was generated in a sub-directory containing a simple TSV format file listing the name of each transcript, its length, effective length, and its abundance in terms of Transcripts Per Million (TPM) as well as estimated number of reads originating from each transcript. After the quantification, transcript counts from Salmon output were imported into R statistical software environment version 4.10 (R Core Team, 2020) and the bootstrap samples were used to estimate the mapping uncertainty for each transcript. Transcripts were then associated with gene IDs for gene-level summarization for downstream analysis.
GRCm39
csv files; meta data and information for each samples.
csv files; raw gene-level counts from summarisation of transcript with the same gene id for each samples.

Submission date

Aug 18, 2023

Last update date

Jul 18, 2024

Contact name

Jonathan Gleadle

E-mail(s)

jonathan.gleadle@flinders.edu.au

Organization name

Flinders University

Department

College of Medicine and Public Health