GSM9430999: HEK 293T wt, rep2; Homo sapiens; OTHER
Multiplex small RNA sequencing (MSR-seq) libraries were prepared as described by Watkins et al. (42), with minor modifications. Total RNA was extracted from wild-type HEK293T and I1174N-9 HEK293T cells using TRIzol (Invitrogen™), and 1 µg of total RNA per sample was used as input. RNA was kept under acidic conditions to preserve aminoacylation status. To preserve tRNA aminoacylation information, only the acylated MSR-seq workflow was performed. Total RNA was subjected to one-pot periodate oxidation and β-elimination to selectively remove the terminal 3' nucleotide from uncharged tRNAs, followed by 3′ end repair with T4 polynucleotide kinase (NEB). Barcoded biotinylated capture hairpin oligonucleotides were ligated to RNA 3′ ends using T4 RNA ligase I (NEB), after which samples were pooled and immobilized on streptavidin-coated MyOne C1 Dynabeads (ThermoFisher). All subsequent steps were performed on-bead, including dephosphorylation, reverse transcription using SuperScript IV VILO (ThermoFisher) under extended incubation conditions, RNase H (NEB) digestion, periodate oxidation to inactivate unligated adapters, and second adapter ligation. Libraries were PCR-amplified using Q5 high-fidelity DNA polymerase (Q5), size-selected by 10% TBE-PAGE (Invitrogen™) to remove terminal transferase by-products, and purified by gel extraction followed by ethanol precipitation.  Final libraries were sequenced on a NovaSeq X using paired-end sequencing.