title: GSM8325367: D25 bovine embryo, replicate 1, scRNA seq; Bos taurus; RNA-Seq
library_construction_protocol: Day 25 flushed bovine chimeric embryos were cut into small pieces and treated with 2 mg/ml papain (Worthington Biochemical, LS003119), 1 mg/ml DNase I (Roche, 10104159001) and 0.5x Glutamax in 2 ml Hibernate A without calcium (BrainBits, HACA500) at 37°C for 40 min. After digestion, 2 ml DPBS with 2% FBS was added to neutralize the enzyme. The embryos were singularized by pipetting 15 to 20 times. The dissociated cell suspension was passed through a 70 μm strainer and centrifuged at 300g for 5 min at 4°C. The cell pellet was resuspended in 1 ml cold RPMI medium with 2% FBS and subjected to cell sorting. After sorting, the cell pellet was resuspended in DPBS with 2% FBS at 800 cells/μL. The concentrated cells were then loaded for 10x single cell RNA sequencing. Prepared cells were processed for single cell transcriptome analysis using the Chromium Next GEM Single Cell 3' GEM, Library & Gel Bead Kit v3.1 (10x Genomics, Pleasanton, CA, USA).  Cells were diluted to the appropriate concentration per manufacturer recommendations prior to ensure proper loading.  GEM reverse transcription, cDNA amplification and library generation followed manufacturer's protocol (10x Genomics, Pleasanton, CA, USA).