GSM7863714: UNM-S_rep3_default; synthetic construct; RNA-Seq
In vitro constructs were artifically poly-A-tailed using PAP (NEB, M0276S), and cleaned up using the RNEasy MinElute kit (Quiagen,  74204) for curlcake constructs and Zymo Clean and Concentrator kit (Zymo Resarch, R1013). Concentrations were determined using a Qubit 2.0 Fluorometer. For libraries that were not generated as part of this work please refer to the respective publications Direct RNA-seqeuncing libraries were generated using the SQK-RNA002 according to the manufacturer's protocol (version : DRS_9080_v2_revR_14Aug2019-minion). For curlcakes a final library of 800ng war generated (200ng per curlcake construct) while 750ng for the UNM-S libraries was used (250ng per riboswitch). The reverse transcription step was peformed using SuperScript III R(Thermo Fisher Scientific, 18080044) in the case of ac4C, m5U-containing curlcake constructs and UNM-S, and Superscript IV (Thermo Fisher Scientific, 18090010) in the case of m1Ψ. For libraries that were not generated as part of this work please refer to the respective publications.