GSM7184360: HeLa-uninfected-tRNA (rep1); Homo sapiens; ncRNA-Seq
For tRNA-seq, total RNA was extacted with Trizol, deacylated in 50 mM Tris-HCl (pH 9.0) at 37 °C for 45 min and then resolved by 10% polyacrylamide TBE-urea gel (Invitrogen). tRNA (60 to 100 nt) were collected by gel excision and resolved in RNA elution buffer (300 mM NaOAc pH 5.2, 1 mM EDTA, 0.1 U/μL SUPERase In) overnight. After ethanol precipitation, tRNA was treated with demethylase (rtStarTM tRNA-optimized First-Strand cDNA Synthesis Kit, ArrayStar) and cleaned up. 1 μg tRNA was used for library construction. For ribo-seq, ribosome fractions separated by sucrose gradient sedimentation were pooled and digested with E. coli RNase I (Ambion, 750 U per 100 A260 units) by incubation at 4°C for 1 h. SUPERase inhibitor (50 U per 100 U RNase I) was then added into the reaction mixture to stop the digestion. Total RNA was extracted using TRIzol reagent. RNA was separated on a 15% polyacrylamide TBE-urea gel (Invitrogen) and the ribosome protected fractions were excised (25-35 nt). RNA was dephosphorylated. Poly(A) tailing, 5' adenylation, adaptor ligation, and library construction were carried out as described in PMID: 36824937.