title: GSM6249226: RNA-Seq MI_1h_rep3; Mus musculus; RNA-Seq
library_construction_protocol: Total RNA was extracted from the samples and the quality of the RNA was tested. Then, mRNA was purified from the total RNA using oligo dT magnetic beads. Fragmentation of mRNA was performed under high temperature conditions using divalent cations in NEBNext First Strand Synthesis Reaction Buffer (5X). Synthesis of first-strand cDNA using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H) as the template for mRNA. Second strand cDNA synthesis was carried out with DNA polymerase I and RNase H. The remaining overhangs were transformed to blunt-ended by exonuclease/polymerase activity. The 3ʹ end of the DNA fragment was adenylated and then the NEBNext adapters with a hairpin loop structure were attached in preparation for hybridisation. Library fragments were purified using the Ampoule XP system (Beverly Beckman Coulter, USA) to prioritize cDNA fragments of 250-300 bp in length. The cDNA was ligated using 3 μl of USER Enzyme (NEB, USA) for 15 minutes at 37°C using an adapter of the selected size, then used for 5 minutes at 95°C, followed by PCR. PCR was then conducted using Phusion High Fidelity DNA polymerase, universal PCR primers and index (X) Primer. Next, the PCR products were purified (Ampoule XP system) and the library quality was evaluated on the Agilent Bioanalyzer 2100 system.