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Sample GSM3072401

Query DataSets for GSM3072401

Status

Public on Jan 31, 2019

Title

8-cell 112815_02 SOLO

Sample type

SRA

Source name

8-cell embryo

Organism

Macaca mulatta

Characteristics

tissue: embryo
developmental stage: 8-cell embryo
library processing kit: SoLo

Growth protocol

Females were observed daily for signs of vaginal bleeding and the first day of menses was assigned cycle day 1. Beginning on cycle day 1–4 recombinant human FSH (r-hFSH: Organon, West Orange, NJ) was administered (37.5 IU) twice daily, intramuscularly for 7 days total. To obtain in vivo matured (VVM) oocytes, females were given recombinant hCG (1000 IU Ovidrel; Serono, Rockland, MA) on treatment day 8 in addition to the FSH treatment outlined above. COCs were removed at 28 – 30 h following hCG by ultrasound-guided aspiration. Oocytes were retrieved from aspirates. In vivo matured oocytes were rinsed and transferred into TL-PVA medium (37°C) under oil and inseminated according to standard procedure for IVF of rhesus macaque oocytes; this is Day 0 of embryo culture. Semen was collected from male macaques that had been trained for this procedure. Sperm were washed from seminal plasma and resuspended in TL-BSA medium. The next morning (Day 1), oocytes were transferred into 70 μl drops of chemically-defined, protein-free hamster embryo culture medium 9 (HECM-9) without or with relaxin (see below) under oil (37°C) and incubated at 37°C in a humidified atmosphere of 5% CO2, 10% O2 and 85% N2 for 48 hours. At approximately 60 hours post insemination non-cleaved oocytes were again assessed for developmental status. Non-cleaved oocytes exhibiting a polar body and all embryos were classified as having matured to metaphase II of meiosis (MII). Embryos were transferred into 70 μl drops of HECM-9 medium with 5% bovine calf serum (Gemini Bioproducts, West Sacramento, CA) with or without relaxin under mineral oil and incubated as described above. Embryos were transferred to fresh medium every other day until fixed on day 8 post insemination. Beginning on Day 5, embryos were observed with a Nikkon SMZ-2B microscope (Melville, NY) housed in a temperature-controlled isolette daily at 0700 and 1700 hrs for developmental stage.

Extracted molecule

total RNA

Extraction protocol

All cumulus cells were removed by repeatedly pipetting oocytes with a narrow-gauge pipette that was just slightly larger than the oocytes while observing oocytes with a stereomicroscope. Using the PicoPureTM RNA Extraction kit, total RNA was isolated from each sample following the manufacturer protocol, including a DNAse digestion (RNase-Free DNase Set; Qiagen, Hilden, Germany) to remove any contaminating DNA.
SPIA libraries: 100 ng of each RNA sample was processed with the Ovation RNA-Seq System v2 using Ribo-SPIATM Technology (NuGen, San Carlos, CA). After this initial processing, a Covaris-2 sonicator was used to mechanically fragment the cDNA to an average of 300 bp. This was then followed by a brief S1 nuclease digestion as previously described (Head et al., 2011). After bead purification, the cDNA was processed through the Ovation Ultralow DR Multiplex Systems 1-16 (NuGen) for end repair, adaptor ligation and final library amplification.
SoLo libraries: cDNA libraries were produced with Nugen Ovation SoLo RNA-Seq(NuGEN, San Carlos, CA). After bead purification, the cDNA was processed: end repair, adaptor ligation and first round library amplification and purification. Then 20 ng of each library was used for the remainder of library preparation, which included use of InDA-C primers for rRNA depletion, as well as 2nd round library amplification and purification steps. SoLo Custom R1 primer was used instead of a standard Illumina R1 sequencing primer for the sequencing of the libraries.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 4000

Data processing

Illumina Real Time Analysis (RTA) v1.18.64 (SPIA libraries) & v2.7.6 (SoLo libraries)
Hisat2 to the Ensembl rhesus monkey genome (Mmul build v8.1.0, genome annotation relase-91)
Removing "ExAmp" duplicates with distance threshold 2500 using in-house developed Python code
DESeq2 comparisons between groups of libraries
Genome_build: Mmul 8.1.0 (Macaca Mulatta)
Supplementary_files_format_and_content: raw counts

Submission date

Mar 30, 2018

Last update date

Jan 31, 2019

Contact name

Uros Midic

E-mail(s)

uros@msu.edu

Organization name

Michigan State University

Department

Animal Sciences