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Sample GSM3911815

Query DataSets for GSM3911815

Status

Public on Apr 02, 2022

Title

Sample type

SRA

Source name

ovary

Organism

Bos taurus

Characteristics

cell type: Bovine IVM oocytes
treatment: oocytes vitrified in liquid helium with 5.6 M cryoprotective agent concentrations.
tissue: ovary

Treatment protocol

After IVM, the oocytes were treated with hyaluronidase (100 IU/mL) for 1–2 min to eliminate the cumulus cells and then washed several times with DPBS solution.

Growth protocol

Oocytes were placed in the droplet of IVM medium (TCM-199 supplemented with 10% [v/v] FBS, 5 μg/mL LH, 0.5 μg/mL FSH, 100 μM cysteine, and 3% [v/v] follicular fluid) covered with mineral oil and incubated at 38.5°C in an atmosphere containing 5% CO2 at maximum humidity for 22–24 h.

Extracted molecule

total RNA

Extraction protocol

Extractions from single cells were amplified for cDNA with SMARTer Ultra Low Input RNA for Illumina Kit, after which QC was performed with Qubit and Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, US).
Library was constructed in steps: amplification if needed, fragmentation, end repairment, poly-A appending at 3'-end, adapter ligation, and enrichment. These steps were completed with SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (Clontech), Ovation Single Cell RNA-Seq System (Nugen), Ovation Ultralow Library System V2 (Nugen), Agencourt AMPure XP Beads (Beckman), Qubit dsDNA HS Assay Kit (Invitrogen), and Agilent High Sensitivity DNA Kit (Agilent).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

HiSeq X Ten

Data processing

Raw reads were preprocessed with Seqtk to filter out adapter sequences in reads, bases near 3'-end with quality value lower than 20, and reads of length smaller than 25, and reads of ribosome RNA.
Clean reads were aligned to reference genome (ensemble Bos taurus.UMD3.1) by Hisat2 (Ver2.0.4) with default parameters.
Gene expression was quantified and then normalized as FPKM(Fragments Per Kilobase of exon model per Million mapped reads). In this step, fragments of each gene were counted with Stringtie(version 1.3.0) and normalized into trimmed mean of M values (TMM), after which FPKM values of every gene were calculated by Perl scripts.
Genome_build: ensemble Bos taurus.UMD3.1

Submission date

Jun 29, 2019

Last update date

Apr 02, 2022

Contact name

Fan Zhang

E-mail(s)

zhangf523@126.com

Organization name

HeNan university of science and technolgy

Street address

No. 263 Kaiyuan Road, Luolong District