title: GSM8718795: NC3; Homo sapiens; RNA-Seq
library_construction_protocol: RNA sequencing： Total RNA was extracted from human knee cartilage or primary-culture chondrocytes using TRIzol reagent, with RNA quality and quantity assessed using the RNA Nano 6000 Assay Kit and the Bioanalyzer 2100 system. mRNA was isolated from total RNA using poly-T oligo-attached magnetic beads and fragmented with divalent cations in an elevated temperature. cDNA synthesis involved using random hexamer primers and M-MuLV Reverse Transcriptase, followed by RNA degradation with RNaseH and second-strand cDNA synthesis using DNA Polymerase I and dNTP. Blunt ends were formed from overhangs, and adaptors with hairpin loop structures were attached. The library fragments, sized 370~420 bp, were purified using the AMPure XP system, PCR-amplified, and purified again. Library quality was verified using Qubit2.0 Fluorometer and Agilent 2100 bioanalyzer, ensuring an effective concentration above 2nM. Qualified libraries were pooled and sequenced on the Illumina NovaSeq 6000, producing 150bp paired-end reads. Sequencing involved fluorescence-labeled dNTPs and real-time synthesis and detection. Image data were converted into sequence data (reads) using CASAVA. Raw data were processed to remove adapters and low-quality reads, generating clean data for downstream analysis. The reference genome was indexed using Hisat2, and clean reads were aligned to the reference genome using the same tool, benefiting from its ability to generate a splice junction database from the gene model annotation file for enhanced mapping results. • Library Preparation Kit: Illumina TruSeq RNA Sample Preparation Kit was used.  • Steps:  • mRNA was isolated using poly-A selection.  • Fragmentation was performed using divalent cations.  • First- and second-strand cDNA synthesis was conducted using random primers.  • cDNA was end-repaired, A-tailed, and ligated with indexed adapters.  • PCR amplification was conducted for library enrichment.  • Quality Control: Libraries were quantified using a Qubit Fluorometer and validated on an Agilent Bioanalyzer.