title: GSM7675808: NTERT cells- UV 3; Homo sapiens; OTHER
library_construction_protocol: For genome-wide tRNA-charging assay, total RNA were extracted with Trizol reagent according to manufacturer's protocol. RNA were then oxidized by NaIO4 followed by deacylation at high pH. Next RNA were purified using ethanol precipitation and used for library preparation. For genome-wide tRNA-charging assay: The purified tRNA samples were then treated with PNK (M0201, New England BioLabs) to remove the 3'-PO4 group from the uncharged tRNA. Next, 5'-adenylated adaptors carrying unique barcode (5'- PO4-NNNNNXXXXXAGATCGGAAGAGCACACGTCTGAA-ddC-3'; N-random nucleotides, XXXXX- is sample specific pentamer barcode, ddc-dideoxy cytidine) were ligated to the tRNAs using T4 RNA ligase2 truncated KQ (M0351L, New England BioLabs). tRNAs were then ligated to a 5'-adaptor (5'-rGrUrUrCrArGrArGrUrUrCrUrArCrArGrUrCrCrGrArCrGrArUrC-3') followed by reverse transcription using SuperScript IV RT kit (18090050, Invitrogen) and RT primer (5'-AGACGTGTGCTCTTCCGATCT-3'). Next the tRNA library was PCR amplified for 10 cycles by using Illumina multiplex PCR primer (5'-AATGATACGGCGACCACCGAGATCTACACGTTCAGAGTTCTACAGTCCGA-3') and Illumina barcode PCR Primer (5'-CAAGCAGAAGACGGCATACGAGATGCCTAAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3'). PCR products were separated by an 8% non-denaturing polyacrylamide TBE gel (EC6215BOX, Invitrogen) and ~210 nt bands were excised, purified and subjected to HiSeq paired-end Illumina sequencing.