title: GSM9459407: NC_2; Homo sapiens; RNA-Seq
library_construction_protocol: RNA was harvested using Rneasy mini plus kit (Qiagen). 1.3 ug of total RNA was used for the construction of sequencing libraries. The integrity of total RNA from each sample was assessed by agarose gel electrophoresis, and RNA concentration and quality were further evaluated using a NanoDrop ND-1000 spectrophotometer. For each sample, 1–2 μg of total RNA was used for RNA sequencing library construction. mRNA was enriched using the NEBNext® Poly(A) mRNA Magnetic Isolation Module, or alternatively, rRNA was removed using the Ribo-Zero Magnetic Gold Kit. The resulting RNA was then used for library preparation with the KAPA Stranded RNA-Seq Library Prep Kit (Roche). This kit employs a dUTP-based strand-specific library construction strategy, which includes the following steps: (1) fragmentation of mRNA-enriched or rRNA-depleted RNA; (2) reverse transcription to synthesize first-strand cDNA; (3) incorporation of dUTP during second-strand cDNA synthesis; (4) end repair and A-tailing of double-stranded cDNA; (5) ligation of Illumina-specific adapters; and (6) PCR amplification and purification to obtain the final RNA-seq libraries. The constructed libraries were quality-controlled using an Agilent 2100 Bioanalyzer to assess library concentration, fragment size distribution (400–600 bp), and adapter contamination, and were quantified by qPCR. Based on the quantification results and the required sequencing depth, libraries from different samples were pooled and subjected to sequencing.