title: GSM8994174: CES16; Rattus norvegicus; RNA-Seq
library_construction_protocol: RNA was extracted using miRneasy mini kit (QIAgen) and quantified using Qubit Flurometer Samples had RNA integrity numbers of 5.5 – 9.6. RNA was shipped to Novogene Co. Ltd. (USA) for library preparation and sequencing. mRNA was purified from total RNA using poly-T oligo-attached magnetic beads and stranded mRNA-seq libraries were generated and individually barcoded using NEBNext® Ultra Directional RNA Library Prep Kit for Illumina (NEB, USA) following manufacturer's recommendations.  Fragmentation was carried out using divalent cations under elevated temperature in NEB Next First Strand Synthesis Reaction Buffer (5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-) ring, the second-strand synthesis in the NEBNext Ultra II Directional RNA Library prep protocol, the reaction uses a buffer containing dUTP, which allows the incorporation of dUTP in place of dTTP. This process is facilitated by the NEBNext Second Strand Synthesis Reaction Buffer with dUTP mix and the NEBNext Second Strand Synthesis Enzyme Mix. The incorporation of dUTP is essential for strand specificity, as it renders the second-strand cDNA uracil-containing, making it susceptible to degradation during subsequent PCR steps. This selective degradation ensures that only the first-strand cDNA is amplified, preserving strand orientation in the final library. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3' ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. To select cDNA fragments of preferentially 150~200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 µl USER Enzyme (NEB, USA) was used with size-selected, adaptor ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR. PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index Primer and PCR products were purified (AMPure XP system). Library quality was assessed on the Agilent Bioanalyzer 2100 system and sequenced by NovaSeq6000 on an S4 flow cell (PE150 read length).