GSM6734716: sws2_bluecone, scRNAseq; Danio rerio; RNA-Seq
To perform single-cell RNA sequencing (scRNA-seq), FACS-isolated cells were loaded onto the Chromium Single Cell Chip (10x Genomics, USA) according to the manufacturer's protocol. The scRNA-seq libraries were generated using the GemCode Single-Cell Instrument and Single Cell 3' Library and Gel Bread kit v2 and v3 Chip kit (10x Genomics, 120237). Library quantification and quality assessments were performed by Qubit fluorometric assay (Invitrogen) and dsDNA High Sensitivity Assay Kit (AATI, DNF-474-0500). The fragment analyser was performed using the High Sensitivity Large Fragment -50kb Analysis Kit (AATI, DNF-464). The indexed library was tested for quality, and sequenced by the Illumina NovaSeq 6000 sequencer with the S2 flow cell using paired-end 150 x 150 base pairs as the sequencing mode. Sequencing depth was 60K reads per cell. Library was performed according to the manufacter's instructions (single cell 3' v3 protocol, 10x Genomics). Briefly, sorted cells were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.