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Sample GSM4357980

Query DataSets for GSM4357980

Status

Public on Jun 30, 2020

Title

G15S-3

Sample type

SRA

Source name

2-cell stage embryos_G15S

Organism

Mus musculus

Characteristics

strain: ICR
mrna injected: actinG15S-HA-NLS
developmental stage: 2-cell stage
tissue: embryos

Treatment protocol

After insemination, fertilized embryos were collected at 2 hpi for mRNA injection. mRNAs were injected using a piezo manipulator (Prime Tech, Tsukuba, Japan).

Growth protocol

After in vitro fertilization, embryos were cultured in potassium simplex optimized medium (KSOM) at 37°C under 5% CO2 until 2-cell stage (32 hours post insemination: 32 hpi).

Extracted molecule

polyA RNA

Extraction protocol

After the removal of zona pellucida, embryos were washed with PBS containing 0.1% BSA, and then transferred to a 0.2 ml tube containing 9.5 μl of reaction buffer to make a final volume of 10.5 μl. After cell lysis in the buffer, 1 μl of the solution was removed and, instead, 1 μl of the diluted ERCC RNA Spike-In Mix was added (1:40,000 dilution; Thermo Fisher Scientific, 4456740). The lysed RNA solution with the spike-in RNA were subjected to reverse transcription.
The produced cDNA was amplified by PCR with 13 cycles. The amplified cDNA was purified using AMPure XP beads (BECKMAN COULTER, A63882). The purified cDNA was measured on the Bioanalyzer (Agilent) using High Sensitivity DNA Kit (5067-4626) and the normalized volume of DNA was subjected to library preparation using Nextera XT DNA Library Preparation Kit (Illumina, FC-131-1024) by following the vendor’s instruction. The obtained library was quality-checked by Bioanalyzer and quantified using Qubit (Thermo Scientific).
Paired end sequencing (50 bp + 25 bp) was carried out using the Illumina NextSeq system.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Description

G15S_G15S3_R1_val_1-pw1_10-cN10-pq20w10m20
processed data file: gene-FPKM_name-2.xlsx
A pool of 5 embryos at the 2-cell stage were subjected to cDNA synthesis

Data processing

Fastq files from Illumina sequencing were filtered for low quality reads by sliding window trimming (window size 10, <QV20), and low quality bases were trimmed from the ends of the reads (<QV20) using Trimmomatic. Reads less than 20 bases and unpaired reads were removed. Furthermore, adaptor, polyA, polyT and polyG sequences were removed using trim_galore and cutadapt.
The sequencing reads were then mapped to the mouse genome (mm10) using STAR. Reads on annotated genes were counted using featureCounts. FPKM values were calculated from mapped reads by normalizing to total counts and transcript.
Genome_build: mm10
Supplementary_files_format_and_content: tab-delimited text files include FPKM values for each sample

Submission date

Feb 27, 2020

Last update date

Jun 30, 2020

Contact name

Kei Miyamoto

E-mail(s)

[email protected]

Phone

81-92-802-4589

Organization name

Kyushu University