GSM9543171: scRNA-seq of E11 to E13.5 sheep embryos; Ovis aries; RNA-Seq
Embryos were digested and fixed right after flushing to avoid RNA degradation. Embryos were chopped and dissociated into single cells by incubation in TrypLE in 4-well dishes in an orbital shaker at 150 rpm, for 20 min at 38.5 °C. During this time, embryos were pipetted every 3 to 5 minutes and finally washed with ice-cold Euroflush medium (IVM Technologies) to quench TrypLE activity. Cells were filtered through Flowmi 40 µm cell strainers, collected in BSA-coated Falcon tubes and centrifuged at 300 G at 4 °C for 8 min. Then, cells were fixed using the EvercodeTM Cell Fixation kit v2 (ECF2101, Parse Biosciences) following the manufacturer instructions. Briefly, cells were suspended in Cell Prefixation Buffer, stained with Trypan Blue to assess cell viability and counted using a haemocytometer. Then, Cell Fixation Solution was added and cells were incubated on ice for 10 minutes. Next, Cell Permeabilization Solution was added, and after 3 min incubation on ice, Cell Neutralization Buffer was added to block the reaction. Then, cells were centrifuged at 300 G at 4 °C for 8 min, suspended in Cell Buffer and frozen in a Mr. FrostyTM freezing container at -80 °C until libraries preparation. Single-cell RNA sequencing libraries were prepared using the Evercode Whole Transcriptome Mini v2 kit (Parse Biosciences) following the manufacturer's protocol. Cells were barcoded using Parse Biosciences' combinatorial barcoding strategy, and two sublibraries with around 12,000 cells each were prepared for paired-end sequencing.