GSM6597902: AjapCNR, replicate 1, scRNAseq; Apostichopus japonicus; RNA-Seq
Fresh CNR tissue was incubated with 2 mg/ml Papain in HE-Ca solution at 37 °C for 20 min and rinsed with Hibernate E®/B27®/GlutaMAX™ (HEB) medium. The single-cell suspension was filtered through a 40 µm cell strainer.  Cell concentration and viability were determined with a Countess® II automated cell counter and 0.4% trypan blue staining. The cell suspension at a concentration of ~1000 cells/µL and cell viability > 90% was ready for scRNA-seq. Cellular suspensions were loaded on a 10X Genomics GemCode Single-cell instrument that generates single-cell Gel Bead-In-EMlusion (GEMs). Libraries were prepared using the Chromium Next GEM Single Cell 3' Reagent Kits v3.1. Every single cell was coated with unique primers containing (i) an Illumina® R1 sequence (read 1 sequencing primer), (ii) a 16 nt 10x Barcode, (iii) a 10 nt Unique Molecular Identifier (UMI), and (iv) a poly-dT primer sequence. Then, Reversed transcription to cDNA and made an amplification in a Veriti rapid thermal cycler (Applied Biosystems, Foster City, CA, USA).