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Sample GSM4810657

Query DataSets for GSM4810657

Status

Public on Mar 24, 2021

Title

oocyte 46

Sample type

SRA

Source name

S30-3

Organism

Homo sapiens

Characteristics

cell type: oocyte
ID: S30
maturation stage: GV

Treatment protocol

Women were stimulated with highly purified urinary hMG (Menopur®, Ferring, Spain) or follitropin alpha (Gonal®, Merck-Serono, Spain), with daily injections of 150-300 IU (Blazquez et al., 2014). A GnRH antagonist (0.25 mg of Cetrorelix acetate, Cetrotide®, Merck Serono, Spain) was administered daily from day 6 of stimulation (for donors) or from when a follicle of 14 mm or estradiol ≥ 400 pg/ml was detected (for patients) (Olivennes et al., 1996). In the case of donors, when 3 or more follicles of >18 mm of diameter were observed, final oocyte maturation was triggered with 0.2 mg of triptorelin (Decapeptyl®, Ipsen Pharma, Spain). In the case of patients, when 3 follicles of >17 mm of diameter were observed, final oocyte maturation was triggered with 250 µg of alpha-choriogonadotropin (Ovitrelle®, MERCK) or 0.3 mg of triptorelin (Decapeptyl®, Ipsen Pharma, Spain).

Growth protocol

Extracted molecule

total RNA

Extraction protocol

Oocyte retrieval was performed 36h later by ultrasound-guided transvaginal follicular aspiration. Oocytes were denuded 30 minutes after pick-up by exposure to 80 IU/ml hyaluronidase (Hyase-10x, Vitrolife, Sweden) in G-MOPS medium (Vitrolife, Sweden), followed by gentle pipetting. Once denuded, oocytes were scored for polar body presence and immature GV were either processed immediately as GV or further cultured in vitro in 50 μl of G2-PLUS (Vitrolife, Sweden) medium in a humidified atmosphere of 6%CO2/94% at 37ºC for 30 hours, when they were checked again for polar body presence and processed.
Full-length single-cell RNA-seq libraries were prepared using the Smart-seq2 protocol (Picelli et al., 2013) with minor modifications. Briefly, oocytes were dezoned with Pronase (Roche Diagnostics, Spain), and individually placed in 2.3 µl of a lysis buffer containing 0.2% Triton-X100 (T8787, Sigma) and 1 U/µl RNAse inhibitor (N8080119, Applied Biosystem), and stored at -80ºC until use. Reverse transcription was performed using SuperScript II (ThermoFisher Scientific) in the presence of 1 μM oligo-dT30VN (IDT), 1 μM template-switching oligonucleotides (QIAGEN), and 1 M betaine. cDNA was amplified using the KAPA Hifi Hotstart ReadyMix (Kapa Biosystems) and IS PCR primer (IDT), with 20 cycles of amplification. Following purification with Agencourt Ampure XP beads (Beckmann Coulter), product size distribution and quantity were assessed on a Bioanalyzer using a High Sensitivity DNA Kit (Agilent Technologies). A total of 140 pg of the amplified cDNA was fragmented using Nextera XT (Illumina) and amplified with Nextera XT indexes (Illumina). Products of each of the 96-well plate were pooled and purified twice with Agencourt Ampure XP beads (Beckmann Coulter). Final libraries were quantified and checked for fragment size distribution using a Bioanalyzer High Sensitivity DNA Kit (Agilent Technologies). Pooled sequencing of Nextera libraries was carried out using a HiSeq4000 (Illumina) to an average sequencing depth of >1 million reads per cell. Sequencing was carried out as paired-end (PE75) reads with library indexes corresponding to cell barcodes (Unique dual indexing).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 4000

Data processing

Primary data analysis and QC assessment was carried out with the standard Illumina pipeline following the manufacturer's protocol
Smart-seq2 libraries were mapped as paired-end reads using STAR 2.5.4b and RSEM 1.3.0 for the quantification
Genome_build: GRCh38
Supplementary_files_format_and_content: Tab-separated values (TSV) files containing gene expression values as raw counts

Submission date

Sep 30, 2020

Last update date

Mar 24, 2021

Contact name

Paula Nieto

E-mail(s)

[email protected]

Organization name

CNAG

Department

CNAG