title: GSM6757514: 80 dph, replicate 1, scRNAseq; Epinephelus coioides; RNA-Seq
library_construction_protocol: A mixture of 30 gonads at 80 dph was dissociated for single-cell transcriptomics with two repetitions. With the development of the fish, 20 gonad tissue samples at 120 dph and 5 gonad tissue samples at 180 dph were mixed and dissociated for scRNA-seq, too. Briefly, gonadal samples were cut into pieces, then cleaned by 1×Hank's Balanced Salt Solution twice. Next, the samples were centrifuged at 1000×g for 3 min and incubated with 500 U/mL collagenase IV (diluted by DPBS buffer without calcium and magnesium ion) at 37 °C for 2 h. Then the samples were centrifuged at 1000×g for 3 min and followed by a filtration step through a 40 µm strainer (Corning, USA). The cell numbers of the suspensions were calculated by the blood counting chamber, then the cells were stained by 0.4% trypan blue to estimate cell viability. Next, the suspensions (cell viability >85%) were used for further single-cell RNA sequencing (scRNA-seq). ScRNA-seq libraries were constructed by Chromium Next GEM Single Cell 3'Reagent Kits v3.1 (10X Genomics, USA). The prepared suspensions were used to generate single-cell Gel Beads-In-Emulsions (GEMs) by loading onto a 10X Genomics GemCode Single-cell. Upon dissolution of the Single Cell 3' 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 are released and mixed with cell lysate and Master Mix. The GEMs were incubated to produce barcoded, full-length cDNA from poly adenylated mRNA. Then the cDNA was amplified by PCR to generate sufficient mass for library construction. R1 (read 1 primer sequence) was 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 contain 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 began and ended 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.