GSM7501733: EKAB_Spinal Cord_Uninjured_rep2; Danio rerio; RNA-Seq
For snRNA-seq, 30 µl of resuspension solution containing isolated nuclei at a concentration of ~1000 nuclei/µl was submitted to sequencing.Two biological replicates of each timepoints at 0, 1, 3 and 6 wpi 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.