GSM8552366: S2_L002, 120 day, scRNASEQ, pig retinal organoid; Sus scrofa; RNA-Seq
At each designated timepoint or period, 5 piPSC-ROs from 3 independent differentiations were pooled for scRNA-seq analysis. Porcine ROs were dissociated using the Neural Tissue Dissociation Papain Kit (P) at 37°C for 1 hr and the resulting cell suspension was filtered through a pre-wet 37 µm cell strainer to obtain a single cell suspension. Cells were counted on a Countess 3FL Automated Cell Counter. Libraries were constructed according to the Chromium Single Cell 3' Reagent Kit v3.1 user guide (10x Genomics, Pleasanton, CA). Briefly, cells in single cell suspension were delivered to the University of Wisconsin-Madison Biotechnology Center on ice where the cell concentration and viability were quantified on the Luna FX-7 automated cell counter (Logos Biosystems, Anyang, Gyeonggi Province, South Korea) using acridine orange/propidium iodide stain (Logos Biosystems). The appropriate volume of cells were loaded onto the Single Cell Chip G required for yielding a targeted cell recovery of 10,000 cells. Following the completion of the Chromium run the GEMs were transferred to emulsion safe strip tubes for GEM-RT using an Eppendorf MasterCycler Pro thermocycler (Eppendorf, Hamburg, Germany). Following RT, GEMs were broken, and the pooled single cell cDNA was amplified. Post-cDNA amplified product was purified using SPRIselect (Beckman Coulter, Brea, CA) and quantified on a Bioanalyzer 2100 (Agilent, Santa Clara, CA) using the High Sensitivity DNA kit. Adapters were then added to the libraries after fragmentation, end repair, A-tailing, and double-sided size selection using SPRIselect. Following adapter ligation, libraries were purified using SPRIselect and sample-specific indexes (Dual Index Plate TT set A, 10x Genomics) were added by sample index PCR. After sample index PCR, samples were double-size selected using SPRIselect yielding final libraries compatible for Illumina sequencing. Final libraries were quantified using the Qubit High Sensitivity DNA Kit (Thermo Fisher Scientific).