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Sample GSM4964796

Query DataSets for GSM4964796

Status

Public on Jun 15, 2022

Title

OM, Embryo 6, TE

Sample type

SRA

Source name

Trophoblast

Organism

Equus caballus

Characteristics

cell type: Blastocyst
tissue: Trophoblast
maternal age: 14
maternal parity: 3
embryo size: 2063
embryo sex: male

Growth protocol

Saddlebred mares (French Anglo-Arabian and Selle Français breeds) were included in this study. They were raised in the “Institut Français du Cheval et de l’Equitation” experimental farm (Chamberet, France, 45° 34’55.17”N, 1°43’16.29”E, 442m). During the protocol period, mares were managed in herds in natural pastures 24h/day with free access to water. The experiments took place from April, the 1st until May 23rd, 2019. All mares remained healthy during this period. The mares’ estrus period was monitored by ultrasound with a 5MHz trans-rectal transducer. During estrus, ovulation was induced with a single 750-1500 IU IV injection of human chorionic gonadotropin (Chrorulon® 5000, MSD Santé animale) as soon as one ovarian follicle >35mm in diameter together with marked uterine edema were observed. At the same time, mares were artificially inseminated with fresh or refrigerated semen containing at least 1 billion motile spermatozoids from a single fertile stallion. Ovulation was confirmed within the next 48 hours by ultrasonography. Embryos were collected 10 days after the insemination (i.e., 8±0.5 days post-ovulation) by non-surgical uterine lavage using prewarmed (37°C) lactated Ringer’s solution (B.Braun) and EZ-Way Filter (IMV Technologies). All embryos were expanded blastocysts grade I or II according to the embryo classification of McKinnon and Squires. They were washed 4 times in commercially available Embryo holding medium (IMV Technologies) and then cut with a microscalpel under binocular magnifying glass to separate pure trophoblast (TE) and inner cell mass enriched hemi-embryo (ICM).

Extracted molecule

total RNA

Extraction protocol

Each hemi-embryo was transferred to a DNA LoBind tube (Eppendorf) with extraction buffer from PicoPure RNA isolation kit (Applied Biosystems) and incubated for 30min at 42°C prior to storage at -80°C. The extraction of hemi-embryos RNA was performed using the PicoPure RNA isolation kit (Applied Biosystems), including a DNAse treatment, following the manufacturer’s instructions. RNA quality and quantity were obtained with 2100 bioanalyzer system using RNA 6000 Pico or nano kit (Agilent Technologies) according to the manufacturer's instructions. The RNA Integrity Number median was 9.8 (range from 8.7 to 10).
Five nanograms of total RNA were mixed with ERCC spike-in mix (Thermofisher) according to the manufacturer recommendations and then used for re-transcription and amplification using the SMART-Seq V4 ultra low input RNA kit (Clontech) according to the manufacturer recommendations. Nine PCR cycles were performed for each hemi-embryo. cDNA quality was assessed on an Agilent Bioanalyzer 2100, using an Agilent High Sensitivity DNA Kit (Agilent Technologies). Libraries were prepared from 0.15 ng cDNA using the Nextera XT Illumina library preparation kit (Illumina). Libraries were pooled in equimolar proportions and sequenced (Paired-end 50-34 pb) on NextSeq500 instrument, using a NextSeq 500 High Output 75 cycles kit (Illumina).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Description

OM_E6_TE_CH

Data processing

Demultiplexing has been done with bcl2fastq2 version 2.2.18.12 (Illumina) and adapters were trimmed with Cutadapt version 1.15, only reads longer than 10pb were kept.
A de novo assembly was made using StringTie version 2.1.2 following manufacturer instructions to identify XIST in equine embryo samples. The GTF output was aligned on Ensembl 99 EquCab3.0 assembly using IGV version 2.7.2. As XIST position and length on X chromosome was knew (thanks to Pablo Ross) and as XIST is annotated in human, the more similar contig was selected. Y chromosome is not assembly in the EquCab3.0 assembly but a nucleotide sequence of SRY is available on NCBI website. Both sequences were added to FASTA file from Ensembl 99 EquCab3.0 assembly. Both genes were added to GTF from Ensembl 99 EquCab3.0 assembly as both unique chromosome with one gene composed of one transcript.
STAR version 2.6 was used for the alignment (2-pass mapping with GTF file using parameters: --alignSoftClipAtReferenceEnds No --alignEndsType EndToEnd --alignIntronMax 86545 --alignMatesGapMax 86545) on previously modified Ensembl 99 EquCab3.0 assembly. Genes were then counted with featureCounts from Subreads package version 1.6.1 (parameters: -p -B -C -M).
Genome_build: Ensembl 99 EquCab3.0 assembly + de novo XIST sequence + SRY sequence from NCBI
Supplementary_files_format_and_content: Matrix table with raw gene counts for every gene and every sample
Supplementary_files_format_and_content: gtf for de novo XIST gene and SRY gene from NCBI
Supplementary_files_format_and_content: fasta file for SRY gene from NCBI (GenBank: AB004572.1)
Supplementary_files_format_and_content: fasta file for XIST de novo annotated uing StringTie and IGV

Submission date

Dec 08, 2020

Last update date

Jun 15, 2022

Contact name

Emilie Derisoud

E-mail(s)

emilie.derisoud@gmail.com, emilie.derisoud@inrae.fr

Organization name

INRAE

Street address

26 Rue des Prés,