title: GSM9081077: 11dpf, replicate 2, scRNA-seq; Danio rerio; RNA-Seq
library_construction_protocol: At 3, 6, and 11 dpf, zebrafish larvae were anesthetized in the E3 medium, and brain tissues were isolated within 30 min. 1500-2000 brain tissues were isolated from the above zebrafish at the corresponding time point and incubated at 37℃ with tissue digestion solution containing 2 mg/L of papain for about 25 min. Then the tissues were incubated with DMEM containing 5% fetal bovine serum (FBS) to terminate the digestion. Cell suspensions were centrifugated at 500 g for 30 min at 4℃ and resuspended with DMEM medium containing 5% FBS. Then cell suspensions were filtered through 70 μm, 40 μm cell strainer to prepare single cell suspensions. All filtration procedures were performed on ice to preserve cell viability. eGFP positive cells were sorted by FACS aril III and collected with DMEM medium containing 5% FBS. Cellular suspensions were loaded on a 10X Genomics GemCode Single-cell instrument that generates single-cell Gel Bead-In-EMlusion (GEMs). Libraries were generated and sequenced from the cDNAs with Chromium Next GEM Single Cell 3' Reagent Kits v3.1. Upon dissolution of the Gel Bead in a GEM, primers containing (i) an Illumina® R1 sequence (read 1 sequencing primer), (ii) a 16 nt 10x Barcode, (iii) a 10 nt Unique Molecular Identifier (UMI), and (iv) 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. Silane magnetic beads were used to remove leftover biochemical reagents and primers from the post GEM reaction mixture. Full-length, barcoded cDNAs were then amplified by PCR to generate sufficient mass for library construction. R1 (read 1 primer sequence) were added to the molecules during GEM incubation. P5, P7, a sample index, and R2 (read 2 primer sequence) were added during library construction via End Repair, A-tailing, Adaptor Ligation, and PCR. The final libraries contained the P5 and P7 primers used in Illumina bridge amplification. The Single Cell 3' Protocol produced Illumina-ready sequencing libraries. A Single Cell 3' Library comprised standard Illumina paired-end constructs which begin and end with P5 and P7. The Single Cell 3' 16 bp 10x Barcode and 10 bp UMI were encoded in Read 1, while Read 2 was used to sequence the cDNA fragment. Sample index sequences were incorporated as the i7 index read. Read 1 and Read 2 were standard Illumina® sequencing primer sites used in paired-end sequencing.