title: GSM9284462: E. coli, adaptive evolution, 40day, rep3; Escherichia coli str. K-12 substr. MG1655; RNA-Seq
library_construction_protocol: Total RNA was extracted from each sample using Trizol, followed by quantifying RNA concentration and purity using a NanoDrop spectrophotometer. For library construction, ribosomal RNA was removed from total RNA, then precipitated using ethanol (Macklin, Beijing, China). The fragmented RNA served as a template for first-strand cDNA synthesis with random hexamer primers. To ensure strand specificity, second-strand cDNA was synthesized in the presence of deoxyuridine triphosphate (dUTP). Directional libraries were created through steps including end repair, A-tailing, adapter ligation, size selection, PCR amplification, and purification. These libraries were quantified with a Qubit 4 Fluorometer (Thermo Fisher Scientific Inc., USA) and a 7500 Fast Real-Time PCR System (Applied Biosystems, USA), while their size distributions were evaluated on an Agilent 2100 Bioanalyzer.