title: GSM8334031: Zebra Control; Danio rerio; RNA-Seq
library_construction_protocol: The samples were carefully resuspended in a cold, calcium- and magnesium-free solution containing 0.04% BSA/PBS. Cell counting was performed using the LUNA-FX7™ Automated Fluorescence Cell Counter (Logos Biosystems) with acridine orange (AO) and propidium iodide (PI) staining (Logos Biosystems, catalog number F23001). Following this, the cells were processed using the Dead Cells Removal Kit (Miltenyi Biotech, catalog number 130-090-101) and MS columns (Miltenyi Biotech, catalog number 130-042-201) in accordance with the manufacturer's guidelines. Following the guidelines of the 10x Chromium Single Cell 5' v2 protocol (10x Genomics, document number CG000331), Single Cell RNA-seq libraries were generated using the 10x Chromium Controller and the Next Gem Single Cell 5' Reagent v2 kits (10x Genomics, PN-1000244). In brief, the cell suspension (aiming for a recovery of 10,000 cells) was combined with the reverse transcription master mix and loaded along with Single Cell 5′ Gel Beads and Partitioning Oil into a Single Cell K Chip (10x Genomics, PN-1000286) to create single-cell Gel Bead-in-emulsion (GEM) droplets. Within these GEMs, RNA transcripts from single cells were uniquely barcoded and reverse-transcribed. After the GEM-RT reaction, where barcoded full-length cDNA was synthesized from mRNA, the barcoded cDNA molecules were enriched with PCR. For the preparation of the 5' Gene Expression Library, the amplified cDNA underwent a series of steps, including enzymatic fragmentation, end-repair, A-tailing, adapter ligation, and index PCR.