GSM6533904: GBM_VU41; Homo sapiens; ncRNA-Seq
Total RNA was extracted from various tissue samples and SU-DHL-5, SNB-75, HEK293T and hESC cell lines, using mirVana Isolation Kit. For all samples, on-column RNase-free DNase treatment was performed. De-aminoacylation was then performed by incubating total RNA in 0.1 M Tris-HCL pH 9.0 and 1 mM EDTA for 30 minutes at 37 °C. Demethylation reaction was performed on total RNA. Briefly, 2 ug total RNA was treated with 19 uM of AlkB consisting of 8.5uM AlkB WT and 10.5 uM AlkB-D135S mutant, 10 mM KCl, 2 mM MgCl2, 283 uM freshly prepared (NH4)2Fe(SO4)2 6H2O, 0,3 mM 2-ketoglutarate, 2 mM freshly made L-ascorbic acid, 40U RNase Inhibitor and 50 mM MES buffer pH 5.0 in a total volume of 40 ul. Reactions were incubated at 25°C for 2h and quenched with 5 mM EDTA. RNA was recovered by an Oligo Clean&Concentrator-5 kit and eluted in 6 ul Nuclease-free water. Native total RNA or demethylated total RNA was used for each cell line and tissue sample. For the workflow of ALL-tRNAseq a 5' phosphorylated 3' end adapter with 4 randomized nucleotides at the 5' end and a 3' blocking group (3C Spacer; 3SpC3, IDT) was adenylated by Mth RNA ligase and then ligated to the RNA template using a T4 RNA ligase 2, truncated KQ for 1 h at 25°C in the presence of 20% PEG. Ligated RNA was gel-purified and size-fractionated on a Novex TBE-urea 10% denaturing polyacrylamide gel to enrich small RNA molecules using two FAM-markers of 25 nt and 39 nt and an upper marker of the low range ssRNA ladder at 150 nt. Gels were stained using 1x SYBR gold nucleic acid stain in 1x TBE for 10 minutes and 3'adapter-ligated-RNA in size range of 39-150 nt were excised on a blue light transilluminator. RNA was recovered from excised polyacrylamide gel pieces by crushing the gel fragments and soaking them in 0.3M NaCl overnight at 4°C and subsequent ethanol precipitation, 5' adapters with 4 terminal, randomized nucleotides at 3' end were added, using T4 RNA ligase and ATP. Ligation was performed for 1h at 25°C in the presence of 20% PEG. Thereafter, reverse transcription was performed for 1h at 50°C or 42°C using SuperScript III or MarathonRT, respectively. Reactions were terminated by incubation with 0,25 M NaOH, at 95°C for 3 min, then neutralized with 0,25 M HCl. Next, cDNA was purified using MinElute Reaction Cleanup kit and PCR amplified using Phusion high-fidelity PCR master mix for 12 cycles of 98°C for 5 s, 62°C for 10s and 72°C for 10 s. A 6-nt index sequences was introduced into the adapter sequence to enable library identification from a sequenced pool. PCR products were equimolarly pooled for cluster generation without additional size-selection. Sequencing of the ALL-tRNAseq libraries was performed as single-end reads for 150 cycles on NovaSeq 6000.