title: GSM9034429: larval trunk, 2dpi Negative Control; Danio rerio; RNA-Seq
library_construction_protocol: 15 larvae per cohort were dissected to remove the head, caudal fin, and internal organs and then processed as described (Farnsworth DB 459 2020). For tissue lysis, 500μL of dissociation buffer (2ug/ul Collagenase P, 2mM CaCl2, 0.2 ul DNaseI, 0.25% Trypsin, in 1X PBS) was added to the samples and incubated at 28 °C until samples were visually dissociated (~15min). Dissociation was stopped with equal volume of stop buffer (10% FBS, 0.001mM EDTA, in 1X PBS). Samples were centrifuged at 750rcf for 7min at 4°C, the supernatant was discarded, and the pellet was resuspended in 100μL of resuspension buffer (1% FBS, 2mM CaCl2, 1X Penicillin/Streptomycin, in DMEM). The suspension was strained through 20μm cell strainer (Pluriselect USA, 43-10020- 40), centrifuged once (500rcf, 1 min, 4°C) to pass cells through the strainer, and centrifuged a second time (750rcf, 7 min, 4°C) to pellet the cells. The cells were resuspended in 100μL of resuspension buffer, centrifugated (750rcf, 7min, 4°C), and the pellet was resuspended in 50μL 0.04% BSA in PBS. 5μl of cells were then combined with 5μl of Hoechst (Invitrogen, H3570), incubated for 10min, and transferred to a hemocytometer (Bulldogbio, NC1731934) to determine cell concentration. An additional 5μl of cells were incubated with Trypan blue (Sigma, T8154) for 5min, transferred to a hemocytometer, and counted to assess cell viability. Samples with > 70% viability were then submitted for single cell sequencing. 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.