GSM6267523: km, cmyb_pu.1, replicate 1; Danio rerio; RNA-Seq
Kidney marrow were isolated from 1-year-old fish, placed in PBS with 5% fetal bovine serum, triturated by pipet, and filtered through a 40-μm cell strainer. The cells were counted and the proportion of living cells was calculated. After ensuring that the proportion of living cells was ≥90%, the cell concentration was adjusted to 1 000 cells/μL. Libraries were generated and sequenced from the cDNAs with Chromium Next GEM Single Cell 3' Reagent Kits v3.1. Briefly, blood 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.