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Sample GSM6588590

Query DataSets for GSM6588590

Status

Public on Nov 15, 2022

Title

Control_#4 Kidney

Sample type

SRA

Source name

~Day 105 fetal kidney

Organism

Bos indicus x Bos taurus

Characteristics

conception method: Artificial insemination
developmental stage: ~D105 fetus
disease status: Healthy

Treatment protocol

Refers to Chen et al. 2013 (PMID: 23751783 and GEO: GSE63509).

Growth protocol

Refers to Chen et al. 2013 (PMID: 23751783 and GEO: GSE63509).

Extracted molecule

total RNA

Extraction protocol

Total RNA was isolated using TRIzol™ Reagent (Invitrogen, 15596026) following the manufacturer’s instructions. The concentration of RNA samples was measured by using the NanoDrop® ND-1000 Spectrophotometer. RNA samples were stored at -80°C.
This RNA-seq was conducted by Novogene (Sacramento, CA, USA). Briefly, total RNA samples were shipped on dry ice to Novogene overnight. Quality controls were performed for all samples using agarose gel electrophoresis to test RNA degradation and potential contamination and using Agilent 2100 Bioanalyzer to check RNA integrity and quantitation. All samples showed RNA integrity numbers (RIN) of 7.5 to 9.1 and passed the quality control. For strand specific sequencing library preparation, mRNA was enriched using oligo(dT) beads and fragmented randomly using fragmentation buffer. cDNA was synthesized by using mRNA template and random hexamers primer, after which a custom second-strand synthesis buffer (Illumina), dNTPs (dUTP instead of dTTP), RNase H and DNA polymerase I were added to initiate the second-strand synthesis. Then, double-stranded cDNA was subjected to terminal repair, 3’ adenylation, sequencing adaptor ligation, uracil-DNA glycosylase degradation, size selection for 250-300bp insert, and PCR enrichment. Quality controls were performed for all libraries including Qubit concentration measurement, testing insert size using Agilent 2100 Bioanalyzer, and quantifying effective concentration using qPCR. Qualified libraries were pooled and sequenced on Illumina HiSeq platform for 150bp paired end reads.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 4000

Description

Original fetal ID: C008

Data processing

Quality of raw reads was assessed using FastQC version 0.11.7 and SolexaQA++ version 3.1.7 dynamictrim function was used to trim low quality reads (Phred < 20). The lengthsort function of SolexaQA++ was used to remove trimmed reads less than 30 bases in length. Hisat2-build was used to create an index of the reference genome, ARS-UCD1.2, and hisat2 version 2.1.0 was used for alignment of the paired end reads to the reference genome with –dta-cufflinks option. Samtools sort was used to sort bam files based on position. Featurecounts was used for read count estimation with read counting performed at a gene feature level (parameter -t gene). EdgeR v3.24.3 was used for differential expression analysis, using the trimmed mean of M values (TMM) method for normalization and a likelihood ratio test to identify differentially expressed genes. Only genes with at least 5 counts per million in all of the control-AI, or all of the ART-normal, or at least 2 of the ART-LOS were used for differential expression analysis. Genes with a false discovery rate (FDR) ≤ 0.05 were considered significant.
Assembly: ARS-UCD1.2
Supplementary files format and content: Plain text files contain raw read count and edgeR output for annotated genes.

Submission date

Sep 16, 2022

Last update date

Nov 15, 2022

Contact name

Rocío Melissa Rivera

E-mail(s)

riverarm@missouri.edu

Organization name

University of Missouri, Columbia

Department

Division of Animal Sciences