GSM7678239: Group1_Introns_Replicate2_denat; synthetic construct; RNA-Seq
Linear mRNA was synthesized using the HiScribe T7 High Yield RNA Synthesis Kit (NEB) with the following modifications: CleanCap AG (3' OMe) (Trilink-7413) was added to a final concentration of 4mM and m1ΨTP (Trilink-1081) was fully substituted for UTP. For linear RNA samples that were injected into mice, they were further purified using POROSTM Oligo (dT)25 affinity resin (Thermo Fisher) according to the manufacturer's instructions. We fragmented all the RNA in MAP fragmentation buffer (18mM MgCl2, 450mM KCl, 300mM Tris HCl pH 8.3) at 95°C for 1 minutes 45 seconds and followed by RNA purification using RNA Clean & Concentrator-5 (R1014, Zymo) according to manufacturer's instructions. We then performed library preparation following SHAPE-MaP protocol to generate cDNA libraries compatible for Illumina sequencing