title: MinION sequencing
design: RNAs for direct RNA sequencing were treated with T4 Polynucleotide Kinase (NEB, M0201) following the manufacturer's non-radioactive phosphorylation protocol to dephosphorylate 3� termini, removing blocked ends that would inhibit efficient polyadenylation. After reaction clean-up using ZYMO RNA Clean & Concentrator-5 kit (ZYMO, R1013), RNAs were polyadenylated using the E. coli poly(A) polymerase (NEB, M0276) following a recently published protocol (Yan et al. 2018). Briefly, 5 �g RNA, 20 units poly(A) polymerase, 2 �l reaction buffer and 1 mM ATP were incubated for 15 min at 37�C in a total reaction volume of 50 �l. To stop the reaction and to remove the enzyme, the poly(A)-tailed RNA was purified using ZYMO RNA Clean & Concentrator-5 kit (ZYMO, R1013). Libraries for direct RNA nanopore sequencing were prepared from poly(A)-tailed RNAs according to the SQK-RNA002 Kit protocol (Oxford Nanopore, Version: DRS_9080_v2_revS_14Aug2019) with minor modifications. Briefly, Agencourt AMPure XP magnetic beads (Beckman Coulter) in combination with 1 �l of RiboGuard RNase Inhibitor (Lucigen) were used instead of the recommended Agencourt RNAclean XP beads to purify samples after enzymatic reactions. Also, the RTA adapter was replaced by custom adapters described in https://github.com/Psy-Fer/deeplexicon to barcode the samples (Smith et al. 2020).  RNA was linearized using Maxima RT H Minus enzyme (Thermo Fisher Scientific,  #EP0751)  and  incubated  for  30  minutes  at  60�C  followed  by  a  heat inactivation step of 5 minutes at 85�C. After RT reactions, cDNA was quantified using the Qubit DNA HS assay kit (Thermo Fisher Scientific) and equimolar amounts of DNA were used for ligation of the RNA Adapter (RMX) in a single tube. Subsequent reactions were performed according to the protocols recommended by Oxford Nanopore Technologies. Finally, libraries were loaded onto R9.4 flow cells (Oxford Nanopore Technologies) and sequenced on a MinION device for 48 h.