GSM8869795: agu79_Family232_48hpf_noc2l+/+_5; Danio rerio; RNA-Seq
At each developmental stage (24 hpf, 36 hpf, and 48 hpf), 6-9 embryos were individually placed into 1.5 mL tubes. To each tube, 200 µL of TRI Reagent-LS was added and the contents were vortexed. Subsequently, 40 µL of chloroform was added, followed by vortexing, and the tubes were left to stand at room temperature for 15 minutes to allow phase separation into an aqueous phase, interphase, and organic phase. The aqueous phase, which contains RNA, was transferred to a new 1.5 mL tube, mixed by inversion with 100 µL of isopropanol, and left to stand for 10 minutes to precipitate the RNA. After centrifugation (12,000 × g, 2°C, 10 minutes), the supernatant was removed, and the RNA was recovered as a pellet. The pellet was washed twice with 75% ethanol and eluted in 10 µL of nuclease-free water (NFW; Cosmo Bio Co., Ltd.). 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).