Ion Torrent Proton sequencing; GSM2318909: Day 10 LE_01; Ovis aries; RNA-Seq

Day 10 LE_01

RNA seq analysis of laser capture microdissected luminal and glandular epithelium from ewes on day of pregnancy 10, 12, 14, 16 and 20. As well as RNA seq of whole conceptuses, and trophectoderm tissue from day 12, 14, 16 and 20 of pregnancy. Determination of gene expression changes in the uterine epithelium and conceptus during early pregnancy helps to improve our understanding of early pregnancy events and provides a basis of new strategies to improve fertility and reproductive efficiency in ruminants. Overall design: RNA seq analysis of 4 samples of each tissue type (luminal epithelium (LE), glandular epithelium (GE) and conceptus) for 4 animals. Pre-sequencing amplification of LE, GE and day 12 conceptus samples.

Roadmap to implantation- RNA seq of ovine LE, GE and conceptus during early pregnancy

Submitted by Gene Expression Omnibus on 18-SEP-2016

Uteri frozen in OCT were cryosectioned (10 µm) using a Leica CM1950 cryostat (Leica Microsystems, Wetzlar, Germany). Three tissue sections were mounted per slide to RNase-free polyethylene naphthalate-coated slides (Carl Zeiss, Munich, Germany) and immediately placed on dry ice. Slides were fixed and stained immediately following sectioning using previously described methods. Briefly, slides were transferred from dry ice into ice-cold 95% ethanol for 30 sec, then washed in 75% ethanol for 30 sec. Tissue sections were stained in freshly filtered 1% cresyl violet solution in 75% ethanol for 1 min. Tissue sections were then dehydrated through 75% ethanol (30 sec) and 95% ethanol (30 sec), followed by two 30 sec and one 5 min incubation in anhydrous 100% ethanol. Slides were dried for 10 min at room temperature and stored in sealed containers at -80ºC until use. The LE and GE were visually identified and separately captured from a single slide of three uterine sections for each animal. Slides were discarded after 60 min of use on the laser capture microdissection (LCM) LMD7 microscope (Leica Microsystems). Total RNA was extracted from cells collected from the LE and GE using the RNeasy MinElute kit (Qiagen, Valencia, CA). Prior to sequencing, 5 ng of total RNA was amplified using the Ovation RNA-Seq System V2 (cat. # 7102; Nugen, San Carlos, CA). The resulting single primer isothermal amplified (SPIA) cDNA was cleaned using the QIAquick PCR Purification Kit (Qiagen). Day 12 conceptuses: Total RNA was extracted from day 12 conceptuses (n = 5) using the RNeasy plus kit with on column DNase digestion (Qiagen). Total RNA quality and quantity was determined using a RNA 6000 Pico Kit and Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA). A total of 374-512 ng of RNA was collected from each conceptus, for each animal. Prior to sequencing, 50 ng of total RNA was amplified using the Ovation RNA-Seq System V2 (Nugen). Days 14, 16 and 20 conceptuses: Total RNA was extracted from conceptuses (n = 5 per day) and quality checked as described for day 12 conceptuses. RNA samples were enriched for mRNA by selective depletion of ribosomal RNA using RiboMinus Eukaryote Kit for RNA-Seq (cat. # A1083708, Thermo Fisher Scientific Inc., Wilmington, DE). LE and GE: The Ion Plus Fragment library kit (Life Technologies, Waltham, MA) was used to construct Ion Proton sequencing libraries from SPIA LCM cDNA (200 ng). Sonication (Bioruptor, Diagenode Inc., Denville, NJ) was used to fragment the cDNA. End repaired cDNA was purified using 1.0X v/v AMPure XP beads (Beckman-Coulter Genomics, Danvers, MA). Following barcode adapter ligation and 7 cycles of amplification, the final libraries were size selected by capturing the fragments bound between 0.7X and 1.0X v/v AMPure XP beads. Emulsion PCR was performed on an Ion Chef instrument using the Ion P1 Hi-Q reagents. Sequencing beads were quantified and evaluated by flow cytometry using a Guava Easy Cyte (Millipore, Billerica, MA) with Sybr Gold (Molecular Bioprobes, Eugene, OR) before loading on an Ion P1 v3 semiconductor sequencing chip. Libraries were sequenced on an Ion Proton using Ion P1 Hi-Q sequencing reagents and 520 flow cells (Life Technologies) by the Washington State University Molecular Biology and Genomics Core. Three barcoded libraries were pooled per P1 chip for sequencing. Base calling and primary analysis was performed using Torrent Suite 4.6. Day 12 conceptus: Following amplification, libraries were constructed and sequenced at the University of Missouri DNA Core following the manufacturer’s protocol with reagents supplied in Illumina’s TruSeq DNA PCR-Free sample preparation kit (cat. # FC-121-3001). Briefly, cDNA (0.5 µg) was sheared using standard Covaris methods (Covaris, Inc., Woburn, MA) to generate average fragmented sizes of 350 bp. The resulting 3’ and 5’ overhangs were converted to blunt ends by an end repair reaction which uses a 3’ to 5’ exonuclease activity and polymerase activity. A single adenosine nucleotide was added to the 3’ end of the blunt fragments followed by the ligation of Illumina indexed paired-end adapters. The adaptor ligated library was purified twice with sample purification beads and quantified using the KAPA library quantification kit (cat. # KK4824; Kapa Biosystems, Inc., Wilmington, MA) and library fragment size confirmed on the Fragment Analyzer (cat. # DNF-472; Advanced Analytical Technologies, Inc., Ankeny, IA). Libraries were diluted and sequenced as 100 bp paired-end sequences on the Illumina HiSeq 2500 platform. Day 14, 16 and 20 conceptus:Libraries were prepared and sequenced by Global Biologics LLC (Columbia, MO, USA). Ribosome-depleted RNA was used as input for the Illumina TruSeq Stranded mRNA HT library construction procedure (San Diego, CA, USA). Prior to sequencing, RNA was fragmented and RT primers were hybridized for cDNA synthesis. The resulting cDNA was prepared for sequencing by 3’ adenylation, adapter ligation, and PCR amplification (98ºC for 30 sec; 14 cycles of 98ºC for 10 sec, 60ºC for 30 sec, 72ºC for 30 sec; 72ºC for 5 minutes). Library validation was performed using a Fragment Analyzer (Advanced Analytical) with HiSens NGS reagents followed by quantitation using the Qubit HS DNA assay (cat. # Q32851; Thermo Fisher Scientific, Inc.) and qPCR library kit for Illumina platforms (Kappa Biosystems, Inc.). Libraries were pooled and sequenced as 100 bp paired-end sequences on the Illumina HiSeq 2500 platform.

Roadmap to implantation- RNA seq of ovine LE, GE and conceptus during early pregnancy

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