title: GSM8666729: Brain, normal sleep, ZT6, rep3; Mus musculus; RNA-Seq
library_construction_protocol: For the brain sample collection, 6 hours sleep deprivations during ZT0 – ZT6 was performed by a gentle handling after 2 weeks of acclimatization. The samples were flash-frozen in liquid nitrogen and stored at -80°C until use. Total RNA was extracted using Direst-zol RNA miniprep (ZYMO RESEARCH, R2052) from the minced cerebral cortex tissues. messenger RNA (mRNA) was isolated using NEBNext Poly(A) Magnetic Isolation Module (NEB, E7490L) according to the manufacture's instruction. RNA-seq libraries were prepared using NEBNext Ultra II RNA Library Prep Kit for Illumina (NEB, E7770). End repair was performed using NEBNext Ultra II End Repair/dA-Tailing Module (New England BioLabs, E7546L) according to the manufacture's instructions. Then, the samples were ligated to the NEBNext Adaptors (New England BioLabs, E6440L) and cleaned up using 1.8X (v/v) SPRIselect beads (Beckman Coulter, B23318). Adaptor-ligated DNA fragments were amplified using NEBNext Unique Dual Index Primer Pairs (New England BioLabs, E6441A) with NEBNext Ultra II Q5 Master Mix (New England BioLabs, M0544S). PCR was performed using the following parameters: initial denaturation at 98°C for 30sec, 7 cycles of denaturation at 98°C for 10sec and annealing/extension at 65°C for 75sec, final extension at 65°C for 5min. Size selections for 150-500bp were performed on the PCR products by applying 0.9X and 1.2X SPRIselect beads successively. The libraries were quantified by qPCR using NEBNext Library Quant Kit for Illumina (NEB, E7642S) according to the manufacture's instruction, and mixed in equal weight proportion to form the sequence library.