title: GSM8791358: TLDR-Seq_HeLa_libamp_R10_bc2; Homo sapiens; RNA-Seq
library_construction_protocol: RNA was harvested using the TRIzol Plus RNA purification kit and Phasemaker tubes Complete system, DNaseI treated and purified using the Purelink RNA Mini kit. RNA quality is assessed on the RNA Bioanalyzer. 8ug of RNA was used as an input per sample for the TLDR-sequencing library prep TLDR-seq library prep was carried out as as follows; Input RNA was dephosphorylated then quantified on the nanodrop. 5 ug of dephosphorylated RNA was used for 3'-end A1 adapter ligation with T4 RNA ligase 2 truncated KQ. Unligated A1 adapters were deadenylated and degraded. cDNA was synthesised using Induro reverse transcriptase, with partially reverse transcribed RNAs digested with RNaseONE. Samples are enriched for m7G capped RNA by cap trapping technology. The cDNA strand of the enriched capped RNA were ligated with barcoded 5'-linkers (A2_bc). The second strand cDNA was generated using LongAmp HotStart Taq 2x Master mix. For amplified samples, PCR amplification was carried out using LongAmp HotStart Taq 2x Master mix. Endprep/dA-tailing and nanopore adapter ligation was carried out using the SQK-LSK114 kit and sequenced on MinIOn flow cells (FLO-MIN114 - R10.4.1)