GSM9451169: AntiAdar-LR-3; Mus musculus; RNA-Seq
Trizol RNA extraction: 1mL trizol added to pelleted cells, dissolved 5min. 200uL chloroform added to trizol treated cells, incubate 2min. Centrifuged at 12 000xg 4C. Aqueous layer treated with 500uL isopropanol, incubate -20C 1hour, centrifuged at 12 000xg 10min 4C, pellet washed with 75% ethanol and centrifuged at 7 500xg 5min 4C. Pellet dried 1min, resuspended in 30uL pure H2O. For the Nanopore direct RNA-seq RNA experiment of the HT-22 anti Adar treatment, samples were pooled from n=4 biological replicates prior to library preparation. Illumina_RNA-seq: Libraries for directional RNA-seq were prepared according to manufacturer's instructions with a size enrichment for longer RNAs >100nt by MiRvana size selection kit preceeding the library construction using NEBNext rRNA depletion kit and NEBNext Ultra RNA Library Prep Kit for Illumina (E7530). Nanopore_Direct_RNA-seq: The libraries were prepared using the vendor recommended protocol for the SQK_RNA002 direct RNA sequencing kit from 1ug of starting material. Briefly, 9.5uL of RNA was mixed with 1uL RTA adaptor (Nanopore), 1.5uL T4 DNA ligase (NEB, M0202M) and 3uL NEBNext quick ligation buffer (NEB, B6058S), and incubated for 15min at room temperature. The first-strand synthesis reaction was constructed from the ligation product by adding 9uL H2O, 2uL dNTP mix (NEB, N0447S), 8uL FS buffer (Invitrogen, 18080093), 4uL 0.1M DTT (Invitrogen, 18080093) and 2uL Superscript III reverse transcriptase (Invitrogen, 18080093) and incubated at 50C for 50min, 70C for 10min, then placed immediately on ice. The sample was then subjected to a 1.8X bead clean up using the vendor recommended protocol with Mag-Bind TotalPure NGS beads (Omega Bio-Tek, M1378), eluting in 23uL H2O. 6uL of the RMX adaptor (Nanopore), 8uL of the NEBNext quick ligation buffer and 3uL of T4 DNA ligase were added to the eluted RNA:DNA hybrid and incubated for another 15min at room temperature, concluded by a 1X bead cleaning reaction. The prepared libraries were loaded and sequenced on a R9.4.1 PromethION flow cell using a PromethION 24 Beta instrument as per manufacturer suggestions.