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Sample GSM5073824

Query DataSets for GSM5073824

Status

Public on May 09, 2022

Title

dGV-2 [dGV_S20]

Sample type

SRA

Source name

Human oocyte at the GV stage that is collected a day after the initial oocyte collection

Organism

Homo sapiens

Characteristics

developmental stage: oocyte at the GV stage

Treatment protocol

The collected and denuded oocytes were incubated in the Sydney IVF fertilization medium (Cook medical) at 37°C under 5% CO2 and 5% O2 in air for in vitro maturation, and oocytes that had matured to the MII stage by 16:00 on the same day of oocyte collection were used for the treatment of in vitro fertilization. The remaining oocytes that had not reached the MII stage by 16:00 were normally discarded, but further cultured for this study. A few hours after the incubation, some oocytes were matured to MII oocytes, and such matured oocytes were subjected to sampling for RNA-seq analyses. At that time, GV and MI oocytes were also sampled. Some remaining oocytes were further cultured in in the Sydney IVF fertilization medium for in vitro maturation overnight and collected one day after for RNA-seq analyses.

Growth protocol

After aspirating follicles with a fine needle under the ovarian echo, the cumulus oocyte complexes (COCs) were picked up under a stereomicroscope. COCs from infertile patients were scheduled for intracytoplasmic sperm injection, and surrounding cumulus cells were removed with recombinant human-derived hyaluronidase (Kitazato) in multipurpose handling medium (MHM; Fujifilm Irvine). After denudation, oocyte morphology was examined and an oocyte at the MII stage was used for infertility treatment. Oocytes other than the MII stage were subjected to in vitro maturation as described in the treatment protocol.

Extracted molecule

polyA RNA

Extraction protocol

Collected surplus oocytes were subjected to micromanipulation to remove their sibling polar bodies, and then zona pellucida was enzymatically removed. 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 (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 16 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

dGV_S20_R1_val_1-pw1_10-cN10-pq20w10m20
Single oocyte was 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 human genome (hg19) 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: hg19
Supplementary_files_format_and_content: tab-delimited text files include FPKM values for each sample

Submission date

Feb 10, 2021

Last update date

May 09, 2022

Contact name

Kei Miyamoto

E-mail(s)

[email protected]

Phone

81-92-802-4589

Organization name

Kyushu University