title: GSM8896937: midbrainOrganoid, PSP, d90, rep2; Homo sapiens; miRNA-Seq
library_construction_protocol: Total RNA was extracted from at least 3 organoids for each replicate using TRIzol reagent according to the manufacturer's instruction Adapters were first ligated to the 3′ and 5′ ends of the small RNA molecules. Following adapter ligation, first-strand complementary DNA (cDNA) synthesis was performed via reverse transcription primer hybridization. The resulting cDNA was then PCR-amplified to generate double-stranded cDNA libraries. Libraries were purified and size-selected to enrich for fragments with insert sizes between 18 and 40 base pairs. Final libraries were prepared for single-end sequencing (SE50, NEBNext® Multiplex Small RNA Library Prep Set for Illumina) with a read length of 50 bases. Library quality and concentration were assessed using a Qubit fluorometer (Thermo Fisher Scientific) and real-time PCR. Fragment size distribution was verified using a Bioanalyzer (Agilent Technologies). Qualified libraries were pooled based on effective concentration and desired sequencing depth, then sequenced on Illumina platforms for downstream analysis.