title: GSM8033009: 76_96hpf_S3; Danio rerio; RNA-Seq
library_construction_protocol: Each stage of  RW zebrafish embryos was dissected and the total RNA was extracted using the RNeasy kit (Qiagen) according to the manufacturer's protocol. 3' mRNA-Seq were conducted according to the Lasy-Seq ver. 1.1 protocol (https://sites.google.com/view/lasy-seq/) (Kamitani et al. 2019; Kashima et al. 2021). Briefly, total RNA was reverse transcribed using an RT primer with index and SuperScript IV reverse transcriptase (Thermo Fisher Scientific, Waltham, MA, USA). Then, all RT mixtures of the samples were pooled and purified using an equal volume of AMpure XP beads (Beckman Coulter, Brea, CA, USA) according to the manufacturer's instructions. Second strand synthesis was conducted on the pooled samples using RnaseH (5 U/μL, Enzymatics, Beverly, MA, USA), and DNA polymerase I (10 U/μL, Enzymatics, Beverly, MA, USA). To avoid the carryover of large amounts of rRNAs, the mixture was subjected to Rnase treatment using Rnase T1 (Thermo Fisher Scientific, Waltham, MA, USA). Then, purification was conducted with a 0.8× volume of Ampure XP beads. Fragmentation, end-repair, and A-tailing were conducted using 5× WGS Fragmentation Mix (Enzymatics, Beverly, MA, USA). The Adapter for Lasy-Seq was ligated using 5× Ligation Mix (Enzymatics, Beverly, MA, USA), and the adapter-ligated DNA was purified twice with a 0.8× volume of Ampure XP beads. After optimisation of PCR cycles for library amplification by qPCR using Evagreen, 20× in water (Biotium, Fremont, CA, USA) and the QuantStudio5 Real-Time PCR System (Applied Biosystems, Waltham, MA, USA), the library was amplified using KAPA HiFi HotStart ReadyMix (KAPA BIOSYSTEMS, Wilmington, MA, USA) on the ProFlex PCR System (Applied Biosystems, Waltham, MA, USA). The amplified library was purified with an equal volume of Ampure XP beads. One microliter of the library was then used for electrophoresis using a Bioanalyzer 2100 with the Agilent High Sensitivity DNA kit (Agilent Technologies, Santa Clara, CA, USA) to check for quality. Then, sequencing of 150-bp paired-end reads was performed using HiSeq X Ten (Illumina, San Diego, CA, USA) by Macrogen, Inc. (Seoul, Korea).