title: GSM8010208: Trmt6 WT2; Mus musculus; ncRNA-Seq
library_construction_protocol: RNA from Drosophila BG3-c2, HEK293T, and human iPS cells was isolated with Trizol (Sigma Aldrich) according to the manufacturer's instructions. For total RNA isolation from yeast, frozen cells were resuspended in 100 mM sodium acetate pH=4.5, 10 mM EDTA pH=8.0, 1% SDS (1 ml per 50 OD600 units). An equal volume of hot acid phenol (pH=4.3) was added, and the cell suspension was vortexed vigorously followed by incubation at 65℃ for 5 min with intermittent mixing. After addition of 1/10 volume 1-Bromo-3-chloropropane (BCP, Sigma Aldrich), samples were centrifuged at 10,000 x g for 5 min and the aqueous phase was transferred to a new tube. Following an additional round of hot acid phenol/BCP and a round of BCP only extraction, RNA was precipitated from the aqueous phase by the addition of 3 volumes of ethanol. For RNA isolation from yeast under conditions that preserve tRNA charging, frozen cells were resuspended in ice-cold 100 mM sodium acetate pH=4.5, 10 mM EDTA pH=8.0. One volume of cold acid phenol (pH=4.3) was added and cells were lysed with glass beads. One-tenth volume of BCP was then added and the samples were centrifuged at 10,000 x g/4℃ for 5 min, followed by a second round of cold phenol-BCP and one round of BCP-only extraction. RNA was ethanol-precipitated from the aqueous phase and pellets were washed in 80% ethanol containing 50 mM sodium acetate, pH=4.5, briefly air-dried, and resuspended in 50 mM sodium acetate pH=4.5, 1 mM EDTA pH=8.0. Two synthetic standards corresponding to E.coli tRNALys(UUU) with intact 3'-CCA (5'-GGGTCGTTAGCTCAGTTGGTAGAGCAGTTGACTTTTAATCAATTGGTCGCAGGTTCGAATCCTGCACGACCCACCA-3') or a 3'-CC (5'- GGGTCGTTAGCTCAGTTGGTAG AGCAGTTGACTTTTAATCAATTGGTCGCAGGTTCGAATCCTGCACGACCCACC-3') were added to 5 - 10 µg of total RNA in a 3:1 molar ratio at 0.06 pmol/µg, followed by incubation at 37oC in 50 mM Tris-HCl pH=9.0 to deacylate tRNAs. Deacylation was omitted for samples subjected to oxidation and β-elimination. Total RNA was subsequently dephosphorylated and purified by ethanol precipitation in 0.3M sodium acetate pH=4.5 with 25 µg glycogen (Ambion) as a carrier. RNA was resolved on a denaturing 10% polyacrylamide/7M urea/1xTBE gel and species migrating at the size range of mature tRNAs (60 – 90 nt) were excised and gel slices were crushed with disposable pestles. Following addition of 400 µl gel elution buffer (0.3M sodium acetate pH=4.5, 0.25% SDS, 1mM EDTA pH=8.0), the gel slurry was incubated at 65℃ for 10 min, snap-frozen on dry ice, and thawed at 65℃ for 5 min. RNA was eluted overnight at room temperature with continuous mixing. Gel pieces were removed with Costar Spin-X centrifuge tube filters and RNA was recovered from the flow-through by ethanol precipitation in the presence of 25 µg of glycogen. 50 to 200 ng of gel-purified tRNA was ligated to one of four adapters with distinct barcodes.Ligation was performed for 3 hours at 25°C in a 20-µl reaction volume containing pre-adenylated adapter and RNA substrate in a 4:1 molar ratio, 1x T4 RNA Ligase Reaction Buffer, 200 U of T4 RNA ligase 2 (truncated KQ; NEB), 25% PEG 8000, and 10 U SUPERase In (Ambion). Ligation products were separated from excess adapter on denaturing 10% polyacrylamide/7M urea/1xTBE gels. Bands migrating at 95-125 nt were excised and ligation products were recovered from crushed gel slices. Reverse transcription reactions contained 125 nM adapter-ligated tRNA pool, 125 nM primer(5'pRNAGATCGGAAGAGCGTCGTGTAGGGAAAGAG/iSp18/GTGACTGGAGTTCAGACGTGTGCTC-3') and 500 nM TGIRT (InGex) in a final volume of 20 µl. Reactions were performed in 50 mM Tris-HCl pH=8.3, 75 mM KCl, 3 mM MgCl2, 5 mM DTT (from a freshly made 100 mM stock), and 10 U SUPERase In. After TGIRT addition, reactions were pre-incubated at 42℃ for 10 min, initiated by addition of dNTPs to a final concentration of 1.25 mM, and incubated at 42℃ for 16 hours in a Themrocycler. For Superscript III RT, template and primer were denatured at 75℃ for 5 min and chilled on ice, and reverse transcription was performed in the presence of 1X First-Strand Buffer, 5 mM DTT, 0.5 mM dNTPs, 10 U SUPERase In, and 200 U Superscript III (Invitrogen) at 57℃ for 60 min. Template RNA was hydrolyzed by the addition of 1 µl 5M NaOH and incubation at 95℃ for 3 min. cDNA between 60 and 150 nt was excised and cDNA was eluted from crushed gel slices in 400 µl 10 mM Tris-HCl pH=8.0, 1 mM EDTA at 70℃/2000 rpm for 1 hour in a Thermoblock, followed by ethanol precipitation. Purified cDNA was circularized with CircLigase ssDNA ligase (Lucigen) in 1x reaction buffer supplemented with 1 mM ATP, 50 mM MgCl2, and 1M betaine for 3 hours at 60℃, followed by enzyme inactivation for 10 min at 80℃. One-fifth of circularized cDNA was directly used for library construction PCR. Amplification was performed with KAPA HiFi Polymerase in 1x GC buffer with initial denaturation at 95℃ for 3 min, followed by five to six cycles of 98℃ for 20 sec, 62℃ for 30 sec, 72℃ for 30 sec. PCR products were purified with DNA Clean&Concentrator (Zymo Research) and resolved on 8% polyacrylamide/1xTBE gels alongside pBR322 DNA-MspI Digest (NEB). The 130-220 bp region of each lane was excised and DNA was eluted from crushed gel slices in 400 µl water with continuous mixing at room temperature overnight. After ethanol precipitation in 0.3M sodium acetate pH=5.5 and 25 µg glycogen, libraries were dissolved in 10 µl 10 mM Tris-HCl pH=8.0, quantified with Qubit, and sequenced for 150 cycles on an Illumina Novaseq platform.