Total RNA was extracted from 6 DIV primary mouse cortical neurons dissected from 3 litters of P0 pups of C57BL/6J mice using RNeasy Plus Mini Kit (Qiagen, 74136) for lncRNA-seq profiling through Novogene Corporation Inc. Firstly, ribosomal RNA was removed from total RNA, followed by ethanol precipitation. After fragmentation, the first strand cDNA was synthesized using random hexamer primers. During the second strand cDNA synthesis, dUTPs were replaced with dTTPs in the reaction buffer. The directional library was ready after end repair, A-tailing, adapter ligation, size selection, USER enzyme digestion, amplification, and purification. The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection. Quantified libraries will be pooled and sequenced on Illumina platforms, according to effective library concentration and data amount required.