GSM8291743: hs_TCIM_Tg_neg_rep2; Danio rerio; RNA-Seq
For tissue lysis, SC tissues were homogenized at a low setting for 15 seconds. Density gradient separation using sucrose solution was used to sediment the nuclei from the supernatant. Final nuclear lysates were resuspended using 100 µl of resuspension solution (1x PBS + 2% BSA + 0.2U/µl RNase inhibitor; New England Biolabs, Cat#: M0314S). Hoechst staining was performed to assess the quality of isolated nuclei based on their morphology. Samples in which more than 70% of the nuclei were scored as 'healthy' were submitted for snRNA-seq. For snRNA-seq, 30 µl of isolated nuclei at a concentration of ~1000 nuclei/µl was submitted to Genome Technology Access Center at McDonnel Genome Institute of Washington University. Two biological replicates of each genotype were used. cDNA was prepared after the GEM generation and barcoding, followed by the GEM-RT reaction and bead cleanup steps.  cDNA was amplified for 11-13 cycles then purified using SPRIselect beads. Purified cDNA samples were then run on a Bioanalyzer to determine the cDNA concentration. GEX libraries were prepared as recommended by the 10x Genomics Chromium Single Cell 3' Reagent Kits User Guide (v3.1 Chemistry Dual Index) with appropriate modifications to the PCR cycles based on the calculated cDNA concentration. For sample preparation on the 10x Genomics platform, the Chromium Next GEM Single Cell 3' Kit v3.1, 16 rxns (PN-1000268), Chromium Next GEM Chip G Single Cell Kit, 48 rxns (PN-1000120), and Dual Index Kit TT Set A, 96 rxns (PN-1000215) were used. The concentration of each library was accurately determined through qPCR utilizing the KAPA library Quantification Kit according to the manufacturer's protocol (KAPA Biosystems/Roche) to produce cluster counts appropriate for the Illumina NovaSeq6000 instrument. Normalized libraries were sequenced on a NovaSeq6000 S4 Flow Cell using the XP workflow and a 50x10x16x150 sequencing recipe according to manufacturer protocol. A median sequencing depth of 50,000 reads/cell was targeted for each Gene Expression Library. After sequencing, the Illumina output was processed using the CellRanger (v6.0.0) recommended pipeline to generate gene-barcode count matrices. A custom reference genome was generated with the “cellranger mkref” command, using the fasta file of zebrafish reference genome GRCz11 constructed from the Ensemble genome build (https://useast.ensembl.org/Danio_rerio/Info/Index)  and the sorted Gene Transfer Format file (v4.3.2) from the improved zebrafish transcriptome annotation (Lawson et al., 2020). Base call files for each sample from Illumina were demultiplexed into FASTQ reads. Then, the “cellranger count” pipeline was used to align sequencing reads in FASTQ files to the custom reference genome. Both exon and intron sequences were aligned. The filtered gene-barcode count matrices generated by “cellranger count” was used for downstream analysis.