ONT Transcriptomic for Mus musculus: testicle
Total RNA was extracted by grinding tissue in TRIzol reagent (TIANGEN)(Animal)/CTAB-LiCl method (Plant)/RNeasy Mini Kit(Qiagen,CA,USA)(cells) on dry ice processed following the protocol provided by the manufacyurer.Quality and quantity of total RNA were evaluated using a NanoDrop One UVVis spectrophotometer (ThermoFisher Scientific, USA) and Qubit 4.0 Fluorometer (ThermoFisher Scientific, USA), respectively.The integrity of total RNA was assessed via a Bioanalyzer 2100 System run using the RNA 6000 Nano Kit (Agilent,Technologies,USA),and only RNAs with RNA integrity numbers (RIN) above 8.0 were used for subsequent treatments and sequencing. The mRNA extraction was conducted with a Dynabeads mRNA Purification Kit(ThermoFisher Scientific, USA). The mRNA was quantified using a Qubit 4 fluorometer and a Qubit RNA HS Assay Kit(ThermoFisher Scientific, USA). The mRNA preparations were either immediately used to prepare a sequencing library or frozen and stored at 80 C until further use.The cDNA-PCR libraries were constructed with the cDNA-PCR Sequencing Kit V14 (Cat# SQK-PCS114, Oxford Nanopore Technologies, Oxford, UK) using 500 ng of total RNA. The cDNA-PCR libraries was loaded to the PromethION Flow cell (FLO-PRO114M),which was performed on the Nanopore PromethION sequencer(Oxford Nanopore Technology, Oxford, UK)at GrandOmics (Wuhan, China) for sequencing.