LeeEtAl2024 libraries
RNA was incubated with 1.25 M single-stranded 3 RNA adapter and 60 U RNA ligase 1 (NEB) in 20 l ligation buffer (100 mM Tris hydrochloride, 50 mM magnesium chloride, 10 mM ATP, 100 mM DTT) at 16C overnight. Ligated RNA was purified with 1.8 vol RNAClean XP beads. Next, cDNA was synthesized by incubating 1 M reverse transcription primer (complementary to the 3 adapter), 500 nM 5 iso-template switch oligo, and 200 U Maxima H Minus Reverse Transcriptase (Thermo Fisher Scientific) at 50C for 2 h, then heated at 85C for 5 min. To remove RNA template, 1 l of RNase A/T1 mix (Thermo Fisher scientific) containing 2 mg/ml of RNase A and 5000 U/ml of RNase T1 was added to the reaction and incubated at 37C for 10 min. cDNA libraries were amplified by PCR with Platinum SuperFi II DNA Polymerase (Thermo Fisher Scientific) as follows: 98C for 30 sec, 14 cycles of 98C for 10 sec, 65C for 30 sec, 72C for 10 min, then 72C for 5 min. Amplified libraries were treated with 20 U exonuclease I (NEB) at 37C for 15 min; the exonuclease was heat inactivated by 80C for 5 min. cDNA libraries were purified using Ampure XP beads and libraries were constructed using the DNA ligation kit (Oxford Nanopore Technologies, SQK-109). Libraries were loaded onto the R9.4.1 flow cell and sequenced using a MinION. Reads were base-called locally using Guppy (Oxford Nanopore Technologies)