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Sample GSM3381794

Query DataSets for GSM3381794

Status

Public on Sep 30, 2019

Title

Intact brain with infection, rep 2

Sample type

SRA

Source name

embryo

Organism

Xenopus laevis

Characteristics

tissue: intact brain
infection: yes

Extracted molecule

total RNA

Extraction protocol

Total RNA for each sample was isolated by using TRIzolTM Reagent (Ambion, Invitrogen #15596018) –chloroform, following standard manufacturer’s protocol.
NGS analyses were performed by The MIT BioMicro Center (Boston, MA). Samples were quality controlled on a Fragment Analyzer (Advanced Analytical) to determine DV200 scores. The mRNA from 1 μg of total RNA was isolated using Illumina human/mouse/rat RiboZero Gold and prepared into Illumina libraries using the Kapa RNA HyperPrep kit and 10 cycles of amplification. Final libraries were pooled and quality controlled using qPCR (Roche LC480II) as well as the Fragment Analyzer and sequenced on HiSeq2000 for 40 nucleotides.
Following sequencing, data processing was performed using the standard Illumina pipeline. Fastq files were generated for data processing and assembly.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

180109Lev_D18-1208_NA_sequence.fastq

Data processing

The quality of the RNA-Seq sequence data was first evaluated using FastQC prior to further downstream analysis. Low quality sequences were trimmed and poor quality reads were removed using Trimmomatic.
Alignments were conducted by Genotypic (India). Eight samples were aligned to the reference based transcriptome Xenopus_laevis_v2, downloaded from NCBI [https://www.ncbi.nlm.nih.gov/genome/?term=xenopus+laevis]. There were two biological replicates per treatment (brain intact, not infected; brain intact infected; brainless, not infected; brainless, infected). The raw data from Illumina was checked for quality using FastQC1 and pre- processed, which included removing the adapter sequences and removing the low quality bases (<q30). Pre-processing of data was done with Cutadapt2. Mapping was performed with HISAT2 to align the high quality data to the reference genome with the default parameters.
Cufflinks was used to estimate and calculate transcript abundance. It results in normalized read count in the form of FPKM values. FPKM is a unit of measuring gene/transcript expression Four comparisons were conducted (1) brain intact NI vs. brain intact UTI, (2) brainless NI vs. brainless UTI (3) brain NI. Cufflinks includes a script called “Cuffmerge” that can be used to merge several Cufflinks assemblies together. The main purpose of this script is to make it easier to make an assembly GTF file suitable for use with Cuffdiff. "Cuffdiff" was used to identify significant changes in transcript expression. Merged GTF files produced by Cuffmerge were used as input in Cuffdiff.
Cuffdiff was used to calculate the differentially expressed transcripts and generated p-values, FDR corrected p-values (Q-value) and the log2fold change values.
Genome_build: X. laevis v9.2
Supplementary_files_format_and_content: Excel sheet with normalized counts for each sample, log2 fold change, p-value, gene identifier.

Submission date

Sep 10, 2018

Last update date

Nov 13, 2022

Contact name

Christopher Martyniuk

E-mail(s)

cmartyn@ufl.edu

Phone

3523173905

Organization name

University of Florida