GSM4154311: Mouse Spleen M6 +AlkB ARM-seq; Mus musculus; ncRNA-Seq
Isolation of total RNA from mouse Spleen tissue was performed using Direct-Zol RNA MiniPrep Kit (Zymo Research) with TRI Reagent (Molecular Research Center, Inc.), typically yielding 400–450 μg of total RNA. Appropriate amounts of TRI Reagents was added to each tissue sample, along with ~400 uL of zirconium silicate beads (1.0 mm), and samples were homoginized using a tissue homoginization centrifuge. Sample size (n = 3) was selected to demonstrate the utility of the protocol at a level of replication achievable by most researchers.30 μg of control or AlkB-treated RNA were processed using the MirVana miRNA Isolation Kit (Life Technologies), according to the manufacturer's instructions, to select for RNA <200 nt. RNA was concentrated to 30 μg using RNA Clean and Concentrate-25 (Zymo Research), and was treated with DNase I (New England BioLabs). Following column cleanup of the RNA, 700ng was used as input for NEBNext Small RNA Library Prep Kit for Illumina (New England BioLabs).  Total Small RNA was isolated from Total RNA samples from each Spleen tissue sample using the miRvana small RNA isolation protocol (Life Technologies). Small RNA samples were divided and treated as minus AlkB control treatment and plus-AlkB experimental treatment. AlkB treatment was performed as previously described (Cozen et al. Nat Methods. 2015). All samples were then treated with T4PNK at pH 6.5 to maximize 3' phosphatase activity for 30 mins. Finally RNA was concentrated after each enzymatic treatment using an RNA clean and concentration-5 kit (Zymo Research). Treated RNA was used for library preparation. For ARM-seq, libraries were constructed as previously described (Hrabeta-Robinson, et al. Methods Mol Biol. 2017) which utilizes the NEBNext Multiplex Small RNA Library Prep Set (NEB).  For RNA-seq, and Panc1 RIP-seq,  NEXTflex™ Rapid Directional qRNA-Seq™ For ARM-seq, 30 μg of control or AlkB-treated RNA were processed using the MirVana miRNA Isolation Kit (Life Technologies), according to the manufacturer's instructions, to select for RNA <200 nt. RNA was concentrated to 2ug using RNA Clean and Concentrate-25 (Zymo Research), and was treated with DNase I (New England BioLabs). Following column cleanup of the RNA, 700ng was used as input for NEBNext Small RNA Library Prep Kit for Illumina (New England BioLabs).