GSM6560511: rb1, scRNAseq; Danio rerio; RNA-Seq
Whole brains were isolated from 3 dpf sibling and rb1smu2/smu2 mutants. Brain tissues were dissociated with 300 μL papain (28 units/mL, Worthington) in papain solution (1% DNase, 12 mg/mL L-cysteine in DMEM/F12), incubated at 37°C for 15 min with proper mix methods. Dissociated cells were washed twice with washing buffer (100 mL: 650 μL 45% glucose, 500 μL 1 M HEPES and 5 mL FBS into 93.85 mL 1×DPBS, sterilized with a 0.22 μm pore size filter), and sterilized with 40 μm cell strainers (BD Falcon) into 300–400 μL washing buffer. Libraries were generated and sequenced from the cDNAs with Chromium Next GEM Single Cell 3' Reagent Kits v3.1. Briefly, cell suspensions were loaded on a 10X Genomics GemCode Single-cell instrument to generates single-cell Gel Bead-In-EMlusion (GEMs). Upon dissolution of the Gel Bead in a GEM, primers containing an Illumina® R1 sequence, a 16 nt 10x Barcode, a 10 nt Unique Molecular Identifier (UMI), and a poly-dT primer sequence were released and mixed with cell lysate and Master Mix. Barcoded, full-length cDNAs were then reverse-transcribed from poly-adenylated mRNA. The pooled barcoded cDNA was then cleaned up with silane magnetic beads, amplified by PCR to generate sufficient mass for library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.