Standard NextSeq 2000 paired-end Sequencing; Single-embryo transcriptomic profiles of mouse blastocysts from mature and young males in the FVB background
Single-embryo transcriptomic profiles of mouse blastocysts from mature and young males in the FVB background
In vitro fertilizations followed a modified protocol (Guan et al. 2014) to increase throughput and synchronize development. Sperm was collected from the cauda epididymis and vasa deferentia of treated males, incubated in HTF capacitation media, and allowed to swim up. Cumulus-oocyte complexes were collected from superovulated females treated with PMSG and hCG in KSOM media, then inseminated with 0.2 million sperm in KSOM + GSH media for four hours in low-oxygen incubator conditions. Zygotes were then washed and cultured to the blastocyst stage under low oxygen (5% CO2, 5% O2 for FVB and B6J backgrounds) or standard oxygen (5% CO2, 20% O2 for B6N background) conditions. ; Single-embryo RNA-sequencing was performed based on the previously published SMART-Seq protocol (Hennig et al, 2018; Picelli et al, 2014; Trombetta et al, 2014) with some modifications. Briefly, embryos were arranged in a 96-well plate, heated briefly, and then subjected to reverse transcription. Following reverse transcription, whole transcript amplification (WTA) was carried out, and cDNA was cleaned up using SPRI beads. The cDNA concentration and quality were verified via BioAnalyzer and Qubit. Tagmentation was, then, performed on each diluted sample using Tn5 transposase preloaded with linker oligonucleotides. Finally, adapter index primers were added in the final PCR. After indexing, samples were pooled, cleaned, and evaluated for concentration and quality using a BioAnalyzer and Qubit.
EMBL
840d2f83-9545-4ae3-8820-faa9a656106e;54eb64a9-4ca1-4a0d-8c55-7ca3625f281b