GSM8068833: KO, scRNAseq; Danio rerio; RNA-Seq
Leukocytes were collected from the intestine samples through Ficoll-Hypaque (1.080 g/mL) density-gradient centrifugation. Briefly,the intestine was carefully excised and transferred through a 40 μm stainless nylon mesh (Greiner Bio-OneGmbH, Germany). The cell suspension was suspended in Leibovitz's L-15 Medium (L-15, Gibco) supplemented with penicillin (100 U/ml, Sigma-Aldrich), streptomycin (100 μg/ml, Sigma-Aldrich) and heparin sodium (10 U/ml, Sigma-Aldrich). The cell suspension was slowly added into a Ficoll-Hypaque (1.080 g/mL) density-gradient centrifugation, centrifuged at 1,200 g for 25 min, and the cell layer of the interface was carefully aspirated and then washed with ice-cold PBS at 400 g for 10 min. Cell quantity and viability were determined using 0.4% trypan blue (Sigma, St. Louis, MO, USA), which showed that more than 95% were living cells. The cells of each group were counted using a cell counting plate. Library was performed according to the manufacter's instructions (single cell 3' v2 protocol, 10x Genomics). Briefly, GCs 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.