title: NextSeq 500 paired end sequencing; RNA-seq to investigate the genetic pathways in brain and melanocyte regeneration using a zebrafish model
design: RNA-seq to investigate the genetic pathways in brain and melanocyte regeneration using a zebrafish model
library_construction_protocol: For stab wound approach, firstly, fish were anesthetized by using Tricaine (Sigma). A 30 gauge syringe needle was pushed through a nostril along the rostrocaudal body axis until the end of the one hemisphere of telencephalon. Then they were allowed for recovery of the wound in fresh water. At 3dpl and 14dpl (day post lesion) their stab lesioned hemispheres were dissected as early and late stage, respectively. They were put into RNAlater solution immediately to prevent tissue degradation. For melanocyte regeneration, Zebrafish melanocytes were chemically ablated by one-day NCP treatment. NCP was washed after 24 hours of treatment. NCP (Sigma-Aldrich) was prepared as 100 mM stock solutions in DMSO. Final concentration of NCP was used as 100 µM for each group. The early and late stages of melanocyte regeneration were determined as 1 day after ablation (dpa) and 7 days after ablation, respectively. Firstly, fish were anesthetized by Tricaine. Once fish were immobilized, the caudal fin of each fish were resected, and fins were placed into QIAzol Lysis Reagent and immediately proceeded to tissue disruption and homogenization. After homogenization of all brain and melanocyte tissues, their RNAs were isolated by RNeasy® Micro Kit (QIAGEN).  The RNA quality and integrity were checked by the Agilent 2100 Bioanalyzer System. Adult zebrafish were fed under normal conditions 1 micromolar Neocuproine for some samples RNA-seq libraries were built using the TruSeq RNA Library Preparation Kit from Illumina