title: GSM9002420: CTRL2_d200_3; Homo sapiens; RNA-Seq
library_construction_protocol: RNA was extracted using the Qiagen MicroRNeasy Kit. The concentration of RNA was measured using the Qubit Fluorometric Quantitation and RNA Broad-Range Assay (Thermo Fisher Scientific, USA). RNA Integrity Number RIN was determined using the Fragment Analyzer 5300 and the Fragment Analyzer RNA kit (Agilent Technologies, USA) and presented a good integrity (RIN>7). For library preparation, the mRNA fraction was enriched using polyA capture from 100ng of total RNA using the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB). Subsequently, libraries were prepared using the NEB Next Ultra II Directional RNA Library Prep Kit for Illumina and NEBNext UDI UMI (NEB, USA) following the manufacturer's instructions. To minimize technical batch effects, library preparations were performed using the liquid handler Biomek i7 (Beckman). The library molarity was determined by measuring the library size (approximately 400 bp) using the Fragment Analyzer 5300 and the Fragment Analyzer DNA HS NGS fragment kit (Agilent Technologies, USA) and the library concentration (>2 ng/µl) using Qubit Fluorometric Quantitation and dsDNA High sensitivity assay (Thermo Fisher Scientific, USA). The libraries were denaturated according to the manufacturer's instructions, diluted to 150 pM and sequenced as paired-end 100bp reads on an Illumina NovaSeqX (Illumina, USA).