title: GSM9344148: APS, 0h, biol rep 2; Gallus gallus; RNA-Seq
library_construction_protocol: Total RNA was extracted using TRIzol (Invitrogen) following phase separation and isopropanol precipitation. Samples stored in 1mL TRIzol at -80C were thawed on ice and homogenized using a cell pestle. Chloroform (200uL per 1mL TRIzol was added, and samples were mixed and incubated at RT for 3min. Following centrifugation at 12,000×g for 15min at 4C, the clear aqueous phase was carefully collected, avoiding the interphase, and transferred to a fresh tube. To precipitate RNA, 0.5uL GlycoBlue (Thermo Fisher Scientific) and 500uL isopropanol were added to each sample. After 10min at RT, samples were centrifuged at 12,000×g for 10min at 4C. The resulting RNA pellet was washed with 1mL of 75% ethanol, vortexed briefly, and centrifuged at 7,500×g for 5min. Ethanol was removed, and pellets were air-dried for 10min before being resuspended in 20uL RNase-free water and incubated at 55C for 10min. RNA integrity was assessed by agarose gel electrophoresis. Approximately 300-500ng of each sample was mixed with loading dye and resolved on a 1% agarose gel in 1× TAE containing GelRed® Nucleic Acid Gel Stain (Biotium). Electrophoresis was carried out at 100V for 1h. Integrity was verified by the presence of distinct 28S and 18S rRNA bands with minimal smearing. Total RNA concentration was measured using the QuBit RNA High Sensitivity kit (ThermoFisher Scientific), and samples were stored at -80C until further processing. Polyadenylated mRNA was enriched from 1ug of total RNA using the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs), following the manufacturer's guidelines. Libraries were constructed using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs). Dual indexing was performed with NEBNext Multiplex Oligos for Illumina Sets 1 and 2 (New England Biolabs). Cleanup steps throughout library preparation were carried out using SPRIselect magnetic beads (Beckman Coulter). Library quality and fragment size distribution were assessed using an Agilent 2100 Bioanalyzer with High Sensitivity DNA Chips and Reagents (Agilent Technologies). Sequencing was conducted on an Illumina NextSeq 2000 platform (P3 flow cell, single-end 50bp reads, roughly 90 million reads per sample) by Cambridge Genomic Services.