GSM9025010: Con F2e Rep 2; Rattus norvegicus; RNA-Seq
On E4.5 dams were sacrificed and embryos flushed from the uterine horns. Blastocysts were cultured in EmbryoLoveTM  media (EmbryoTechTM Laboratories Inc., Haverhill, MA) for 2-4 hours then washed in 1x DPBS with 0.04% BSA. They were transferred to pre-warmed Acidic Tyrode's solution (Sigma-Aldrich, St. Louis, MO) for 1-2 minutes to remove the zona pellucida. Embryos were washed again, then transferred to pre-warmed dissociation media (GibcoTM TrypLETM Select Enzyme, Thermo Fisher Scientific, Waltham, MA) for 10 minutes at 37°C followed by careful trituration by pipetting through a pulled glass capillary tube. Libraries were made using the 10X Genomics scRNA-seq platform. Due to the low number of cells collected from the embryos and the cell number requirements for the 10X platform, the rat cells were combined with 1000 human 293T cells. Specifically, all cells were suspended in 1x DPBS with 0.04% BSA, which was mixed into a reaction containing reverse transcription reagents and loaded onto a Chromium Next GEM Chip G (Catalog number 1000120, 10X Genomics, Pleasanton, CA). Reverse transcription, GEM generation and library preparation were performed using the Chromium Next GEM Single Cell 3' Kit v3.1 (Catalog number 1000268, 10X Genomics, Pleasanton, CA). The 10X Genomics Chromium Controller instrument was used to per-form GEM (Gel beads-in-emulsion) generation. Full-length barcoded cDNAs were pro-duced from poly-adenylated mRNA by reverse transcription within the GEMs. The tran-scripts were recovered from the GEMs, and the cDNAs were purified and amplified using 13 amplification cycles as per manufacturer recommendation. After clean-up of the amplification reaction, the concentration of cDNA solution (1µl) was determined a Bioana-lyzer with the High Sensitivity DNA kit (Agilent, Santa Clara, CA). To prepare sequencing libraries compatible with the Illumina platform, the entire cDNA reaction was fragmented, followed by end repair, A-tailing and adapter ligation. cDNA samples were am-plified using dual sample indexes in the final step of library preparation, which enabled multiplexing during sequencing, then cleaned up, quantified, and sent to an outside ven-dor (Novogene, Durham, NC) for sequencing