title: GSM5587076: HMCs, Per2_WT; Mus musculus; RNA-Seq
library_construction_protocol: Total cellular RNA was isolated using the TRIzol reagent (Life Technologies) and a modified protocol that incorporates an additional extraction with phenol/chloroform/isoamyl alcohol (25:24:1, pH 4.3). Indexed RNA-seq libraries were prepared using the NEBNext Ultra Directional RNA Library Prep Kit (New England BioLabs, Ipswich, MA), according to the manufacturer's standard protocol. Briefly, poly-adenylated mRNA was purified from total RNA (100 ng) and rRNA removed by binding to magnetic oligo(dT)25 beads, which was followed by RNA fragmentation. Double-stranded cDNA was then generated by random-primed first-strand synthesis and subsequent second strand synthesis in the presence of dUTP for strand marking. The double-stranded cDNA was then end repaired, 3'-dA tailed, and an Illumina-compatible NEBNext adaptor was ligated. Uridine excision was performed with the USER (Uracil-Specific Excision Reagent) enzyme, and the libraries were then indexed and enriched by high-fidelity PCR amplification (15 cycles) with Q5 High-Fidelity DNA Polymerase and NEBNext multiplex primers.