title: GSM6674152: 5'-Seq Replicate 3; Saccharomyces cerevisiae; OTHER
library_construction_protocol: Hot phenol RNA extraction. 1 µg mRNA was obtained from DNased, total RNA (from the strain with random sequence artificial chromosome) via oligo d(T)25 magnetic bead enrichment. RNA was fragmented with NEBNext® Magnesium RNA Fragmentation Module and purified with ethanol precipitation. RNA was dephosphorylated with Quick CIP (NEB), heat inactivated and pre-adenylated 3' adapter (Jin et al., 2015) was ligated with concentrated T4 RNA Ligase 1 (NEB). Reaction was split in two – mRNA deccaping enzyme (MDE, NEB) was added to one reaction, the other served as a control. 5' adapter (Jin et al., 2015) was ligated with T4 RNA ligase 2 truncated (NEB), reaction was reverse transcribed, and the library was PCR amplified.