GSM8775367: Well P1-10A; Danio rerio; OTHER
tiny sci sci-RNA-seq protocol Three heads per biological replicate and three biological replicates per genotype were pooled in 100 µl lysis buffer B in DNA LowBind tubes (Eppendorf, 002431021). Heads were homogenized with a tissue homogenizer at 1 second intervals for 30-45 seconds until no clumps remained. Following fixation with ice-cold methanol and dithiobis (succinimidyl propionate) (DSP, Thermo Fisher, 22586), nuclei were resuspended in sucrose PBS TritonX MgCl2 (SPBSTM) and sonicated for 12 seconds on low at 4°C using a Bioruptor® Pico (Diagenode). Nuclei were resuspended in SPBSTM with dNTPs, and 5 ul of each of the twelve samples was pipetted into each well of one column of a twin.tec LoBind PCR plate (Eppendorf, 0030129512). Reverse transcription, ligation, final distribution, second-strand synthesis, protease digestion, tagmentation, PCR amplification, and purification were performed as described except that tagmentation was 10 minutes instead of 5 minutes (Martin et al. 2023). Approximately 1000 nuclei were added to each well during the final distribution step. Plate 1 primer sequences were used for reverse transcription, ligation, and PCR. The library was sequenced on a NextSeq500 by the UAB Heflin Center for Genomic Science Core Laboratories using a 75 cycle kit and 34 cycles for Read1, 10 cycles for Index, and 48 cycles for Read2.