GSM7432186: Single-trmAKO_rep3; Escherichia coli; OTHER
Total RNA was extracted from cell pellets using TRIzol (Invitrogen) using the manufacturer's instructions. In brief, cell pellets were resuspended in 3 mL TRIzol, followed addition of 0.6 mL chloroform. Following centrifugation, the aqueous layer was transferred to a new tube and precipitated with isopropanol. The resulting RNA was washed several times with 70% ethanol and resuspended in water. Total RNA was used to sequence tRNA with MSR-seq as described previously (PMID: 35513407). Briefly,  RNA samples treated to beta elimination to assay for tRNA charging, then RNA was ligated to barcoded adaptors. Then reverse transcription was performed with SSIV overnight. cDNA was ligated to another adaptor followed by indexing PCR and illumina sequencing PMID: 35513407