GSM9393611: RNA_AP_vHPC_6; Mus musculus; RNA-Seq
Total RNA was extracted using TRIzol-chloroform and the RNeasy Mini Kit (Qiagen 74104) with on-column RNase-free DNase (Qiagen 79254) treatment according to manufacturer protocols. Total RNA quality was assessed using the High Sensitivity RNA ScreenTape Assay on a TapeStation 4200 system (Agilent 5067-5579, 5067-5580). mRNA was isolated from total RNA using the NEBNext PolyA mRNA Magnetic Isolation Module (NEB E7490). Libraries were prepared using the NEBNext Ultra II RNA Library Prep Kit (NEB E7770) [CTX, dHPC] or the NEBNext Ultra II Directional RNA Library Prep Kit (NEB E7760) [vHPC, DS, VS] in coordination with compatible NEBNext Multiplex Oligos (NEB E7335, E7500, E7710 [CTX, dHPC]; NEB E6440 [vHPC, DS, VS]) and HighPrep PCR beads (MagBio AC-60050). Library quality was assessed using the DNA ScreenTape Assay on a TapeStation 4200 System (Agilent 5067-5602, 5067-5582). Libraries were quantified using the NEBNext Library Quant Kit (NEB E7630) on an Applied Biosystems QuantStudio 5 Real-Time PCR system, and the concentration of amplifiable templates in each library was calculated using the NEBioCalculator qPCR Library Quantification Web Tool. Libraries were then pooled in equal concentrations and submitted for sequencing.