GSM9500778: amputated 6 h EIPA rep 1; Danio rerio; RNA-Seq
Bulk RNA-sequencing was conducted on three biological replicates per condition, each consisting of pooled tail tissue from 30 wildtype AB larvae treated and amputated on the same days. Tail tissue was isolated by dissection on ice, with amputated tails cut approximately 200 to 250 µM from the wound site and unamputated tails cut at the anterior end of the pigment gap. Tail chunks were placed individually in PCR strip tube caps, and caps were inverted into PCR strip tubes containing 50 µL TRIzol (Invitrogen, 15596026) each. Tissue was partially dissociated by pipetting before TRIzol samples were pooled and further dissociated with a 30G syringe. Samples were vortexed and stored at -80°C. Total RNA was extracted from TRIzol samples using 0.5 volumes chloroform. RNA was precipitated from the resulting aqueous layer overnight at -80°C using 1 volume ice cold isopropanol, 0.1 volumes ice cold 3M sodium acetate (Invitrogen AM9740), and 0.01 volumes Glycoblue (Invitrogen, AM9515). RNA samples were washed 3X with 70% ice cold ethanol and resuspended in 15 µL nuclease-free water. Samples were submitted to Novogene for QC, library preparation by poly-A capture, and 150 bp paired-end sequencing (Illumina X Plus).