GSM4154346: Mouse Brain M4 -AlkB DM-tRNA-seq; Mus musculus; ncRNA-Seq
Isolation of total RNA from mouse Brain 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.50 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 using a RNA Clean and Concentrate-25 (Zymo Research), after an on-column DNase I treatment to remove residual DNA. (New England BioLabs). 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). PCR-amplified libraries were purified using DNA Clean and Concentrate-5 (Zymo Research) and then size-selected (140-250 nts) on a 6% non-denaturing TBE-acrylamide gel. Libraries were eluted from gel pieces using Gel Elution Buffer (NEB) and precipitated using .3 M NaOAc, 80% Ethanol, and 1 uL of Linear Acrylamide (supplied in NEBNext Kit) at final concentration.  Samples were left in -80C freezer overnight to precipitate. Precipitated libraries were then pelleted, washed twice in 80% Ethanol, and resuspended in Pure H2O. Libraries were then quantified using NanoDrop and Agilent DNA High Sensitivity kit.                                                                                                                                                                                                         For DM-tRNA-seq, libraries were constructed as using the TGIRT™ Improved Modular Template-Switching RNA-seq Kit (InGex, LLC), which utilizes Illumina-compatible adapters for amplification and sequencing purposes. Libraries were amplified using PCR primers supplied with the NEBNext Small RNA library Prep Set (NEB) and  purified using Agencourt AMPure XP beads (Beckman). Resulting purified sequencing libraries were quantified using NanoDrop and Agilent DNA High Sensitivity kit. Libraries were then pooled equimolarly, and concentrated using a DNA Clean and Concentrate-5 (Zymo Research).  Pooled PCR-amplified libraries were size-selected (140-250 nts) on a 6% non-denaturing TBE-acrylamide gel to remove unwanted primer dimer products. Libraries were eluted from gel pieces using Gel Elution Buffer (NEB) and precipitated using .3 M NaOAc, 80% Ethanol, and 1 uL of Linear Acrylamide (supplied in NEBNext Kit) at final concentration.  Samples were left in -80C freezer overnight to precipitate. Precipitated libraries wer then pelleted, washed twice in 80% Ethanol, and resuspended in Pure H2O.