title: GSM9342742: High exopher number, biol rep 2; Caenorhabditis elegans; RNA-Seq
library_construction_protocol: Total RNA was extracted using Qiagen RNeasy Plus mini kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. RNA samples were quantified using the Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA), and their integrity was assessed with the Agilent TapeStation 4200 (Agilent Technologies, Palo Alto, CA, USA). RNA sequencing libraries were constructed using the NEBNext Ultra II RNA Library Prep Kit for Illumina, following the manufacturer's protocol (NEB, Ipswich, MA, USA). In brief, mRNA was enriched using Oligo(dT) beads and subsequently fragmented at 94°C for 15 minutes. First and second-strand cDNA synthesis was performed, followed by end repair and adenylation of the cDNA fragments at their 3' ends. Universal adapters were then ligated to the cDNA fragments, and libraries were enriched through a limited-cycle PCR with the addition of indexing sequences. The resulting sequencing libraries were validated using the Agilent TapeStation and quantified with both the Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA, USA) and quantitative PCR (KAPA Biosystems, Wilmington, MA, USA). The libraries were multiplexed and loaded onto the Illumina NovaSeq 6000 platform according to the manufacturer's instructions. Sequencing was performed with a 2×150 paired-end (PE) configuration using v1.5 reagents. Image analysis and base calling were carried out using the NovaSeq Control Software v1.7. The raw sequence data (.bcl files) generated were converted into fastq files and de-multiplexed using the Illumina bcl2fastq software (version 2.20), allowing for one mismatch in index sequence identification