title: GSM8543111: NC_A_3; Rattus norvegicus; RNA-Seq
library_construction_protocol: The cultured cells were examined under a microscope to ensure cell integrity and prevent RNA degradation by RNases released from damaged cells. Cells were then washed twice with pre-chilled 1×PBS to remove impurities. Appropriate amounts of Ambion TRIzol were added to isolate total RNA from each cell sample. For each sample, 1.5 µg of RNA was used as input material, and the NEBNext® Ultra™ RNA Library Prep Kit for Illumina® was employed for library preparation, with index codes added to differentiate samples. mRNA was purified using poly-T oligonucleotide-conjugated magnetic beads, and fragmentation was carried out at high temperatures using the NEBNext First Strand Synthesis Reaction Buffer. First-strand cDNA was synthesized using random hexamer primers and M-MuLV reverse transcriptase, followed by second-strand cDNA synthesis using DNA polymerase I and RNase H. Overhang ends were converted to blunt ends using exonuclease/polymerase activity, followed by adenylation of the 3' ends.NEBNext adapters with hairpin loop structures were then ligated for subsequent hybridization. Selected cDNA fragments of 200-250 bp were purified using the AMPure XP system, followed by treatment with USER enzyme and PCR amplification. The final PCR products were purified again and quality assessed using the Agilent Bioanalyzer 2100 system. Sequencing was performed on the Illumina NovaSeq 6000 platform at Allwegene Technology in Beijing, generating 150 bp paired-end reads. Sequencing data were aligned with the reference genome, and FPKM values were calculated to quantify gene/transcript expression.