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Sample GSM3765249

Query DataSets for GSM3765249

Status

Public on Aug 31, 2019

Title

PP_InVivo_5

Sample type

SRA

Source name

Pregnant at 30 and 60 days of gestation_demi-blastocyst

Organism

Bos taurus

Characteristics

breed: Holstein heifers
pregnancy status: Pregnant at 30 and 60 days of gestation
cell type: Demi-blastocyst containing trophectoderm and inner cell mass cells

Treatment protocol

Blastocysts collected on day 7 of culture were transferred individually into 50 µl drops of embryo biopsy medium (BO-biopsy, IVF Bioscience) placed on the surface of an epoxy-printed well slide to which scratches were made manually in the glass with an 18 gauge needle. Scratches were made to assist in stabilization of the embryo during the biopsy procedure. While visualizing the embryo using an inverted microscope with 20X magnification (Nikon‎,Diaphot, Tokyo, Japan), a micro-blade fixed to a micromanipulator was gently pressed against the middle of the embryo so that trophectoderm and inner cell mass cells were evenly distributed on both sides. The micro-blade was slowly moved forward and backward until the embryo was completely bisected and the zona pellucida was removed. One portion of the bisected blastocyst (the smaller or less intact portion if bisection was not uniform) was directly placed into a 0.2 ml tube containing 50 µl of RNAlater (Invitrogen, Carlsbad, CA, USA) and stored at -80°C until RNA-seq analysis. The other demi-embryo was placed into a 25 µl drop of SOF-BE2 covered in mineral oil and maintained inside an incubator at 38.5o C in a humidified atmosphere of 5% O2, 5% CO2 and 90% N2) for 30 min for re-establishment of the blastocoele after bisection. The demi-embryo was then placed into a plate containing embryo transfer medium (BO-Transfer, IVF Bioscience), loaded into a 0.25 ml embryo transfer straw, and transported to the farm in a portable incubator at 38.5°C (Micro Q Technologies). The embryo was the transferred into a heifer recipient.

Extracted molecule

total RNA

Extraction protocol

Total RNA from each sample was isolated using the RNeasy Micro Kit (Qiagen, Germantown, MD, USA) according to the manufacture’s cell protocol with the following adjustments for samples maintained in RNAlater; 250 µl of 100% ethanol was used instead of the recommended 350 µL of 70% (v/v) ethanol and isolated RNA was eluted in 12 µl RNase-free water instead of the recommended 22 µl RNase-free water.
Barcoded fragment libraries were constructed using the Bovine Custom Any Deplete Ovation SoLo RNA-Seq System from NuGEN (San Carlos, CA, USA) following the manufacturer’s protocol. DNA integrity of the libraries was assessed using the High Sensitivity D1000 ScreenTape on the Agilent 4200 TapeStation (Santa Clara, CA, USA) and DNA concentration was measured using the KAPA qPCR kit (Boston, MA, USA) following the manufacturer’s protocol. Samples were then multiplexed and sequenced with 1×75bp single-end reads on an Illumina NEXT-seq (Illumina, Inc.San Diego, CA, USA) at the Stanford Functional Genomics Facility (Stanford, CA, USA) according to the manufacturer’s protocol.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Description

37_S23

Data processing

Sequence reads for each sample were mapped to the annotated bovine reference genome (UMD3.1.70) using CLC genomics workbench 12.0 (www.qiagenbioinformatics.com)
Gene expression levels were assessed by counting the number of mapped reads for each Ensembl ID.
Raw counts were processed with the edgeR package for the R software.
Genes with low expression counts (less than 1 count per million) were filtered out before normalization.
The between-samples normalization method applied was TMM (weighted trimmed mean of M-values).
Genome_build: bosTau8
Supplementary_files_format_and_content: tab delimited text file containing raw read counts for each sample.

Submission date

May 14, 2019

Last update date

Aug 31, 2019

Contact name

Maria Belen Rabaglino

E-mail(s)

m.b.rabaglino@uu.nl

Organization name

Utrecht University

Department

Population Health Science