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Sample GSM7993378

Query DataSets for GSM7993378

Status

Public on Jun 05, 2024

Title

C57BL/6J_day15_sample2

Sample type

SRA

Source name

Fetal brain

Organism

Mus musculus

Characteristics

tissue: Fetal brain
strain: C57BL/6J
fetal sex: Female

Growth protocol

The inbred mice strains C57BL/6J (Strain # 000664), AKR/J (Strain # 00064) and B6.Cg-Cav1tm1Mls/J (aka Cav1-null, Strain # 007083) used in this study were obtained from the Jackson Laboratory (Bar Harbor, ME USA). Approximately 8-weeks old mice of these strains were used to establish timed pregnancy separately. The vaginal plug was observed to keep a record of the start of pregnancy (day 1). On days (d) 12, 15 and 17, the pregnant mice were euthanized, and the fetuses were collected, washed in sterile PBS and snap frozen in liquid nitrogen. The whole brain was dissected from each fetus. Fetal sex was determined by polymerase chain reaction assay as described earlier.

Extracted molecule

total RNA

Extraction protocol

Total RNA were isolated from the dissected fetal brain samples using an AllPrep DNA/RNA Mini Kit (Qiagen, Cat No./ID: 80204) following the manufacturer’s instruction. A total of 27 samples were used which included 3 strains x 3 time points x 3 replicates.
Messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers followed by the second strand cDNA synthesis. The library was ready after end repair, A-tailing, adapter ligation, size selection, amplification, and purification. The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Description

B5d15

Data processing

The quality of raw sequences was checked with FastQC followed by trimming the adaptors from the sequence reads by cutadapt. The fastp tool was used to perform base quality trimming (Phred score >30) by sliding window scan (4 nucleotides). The quality reads were then mapped to the mouse reference genome GRCm39 using STAR. The read count data was then analyzed by edgeR to determine significance of differential expression.
The multi-sample 2-pass mapping approach of STAR was applied to analyze splice variants in fetal brain of the three strains at each developmental time points.
The alternative splice variants were detected based on the presence or absence of mapped reads to the junction sites in the fetal brain. We defined a developmental time specific splice variant as a transcript which showed evidence of 10 or more reads mapped to the transcript junction in a specific strain at a specific developmental time point (i.e. zero read mapped to the same junction in the other strains and time points).
The genes associated with the detected splice junctions in the individual samples were identified by intersecting the detected junction sites with the exon coordinates of mouse genes by using BEDTools.
Assembly: GRCm39
Supplementary files format and content: CSV file (number of reads mapped to each gene in each sample)
Supplementary files format and content: CSV file (number of reads mapped to each splice junction in each sample). We defined a splice variant as an alternative form of a messenger RNA where 10 or more number of sequence reads mapped to ends (i.e. the acceptor and donor positions) of the splice junction.

Submission date

Dec 26, 2023

Last update date

Jun 05, 2024

Contact name

SUSANTA BEHURA

E-mail(s)

behuras@missouri.edu

Phone

5738821722

Organization name

University of Missouri