| Full name | AlkB-facilitated RNA Methylation sequencing |
| Summary | ARM-Seq uses pre-treatment with Escherichia coli AlkB to demethylate 1-methyladenosine, 3-methylcytidine, and 1-methylguanosine foloowed by tRNA sequencing |
| Key findings | tRNA-derived small RNAs accurately recapitulate the m1A modification state in yeast and human |
| Sequencing strategy | RNA-Seq |
| Raw data | https://www.ncbi.nlm.nih.gov/sra/?term=SRP056032 |
| Experimental protocol | 50 µg of control or AlkB-treated RNA was processed using the MirVana miRNA Isolation Kit (Life Technologies), according to manufacturer’s instructions, to select for RNA < 200nt. RNA was concentrated to 25 µg using RNA Clean and Concentrate-25 (Zymo Research), and 10 µg was treated with DNase I (New England BioLabs). Following column cleanup of the RNA, 1 µg was used as input for NEBNext Small RNA Library Prep Kit for Illumina (New England BioLabs). Libraries were size selected on 2% SizeSelect agarose E-Gels using the 50 bp E-gel ladder (Life Technologies Corporation) as a marker to select for bands corresponding to libraries of RNA between 18–120 nt. Dilutions from column cleaned and concentrated libraries were assessed by BioAnalyzer traces using Agilent High Sensitivity DNA kit (Agilent Technologies) |
| Analysis protocol | Reads were trimmed, removing barcoding indices and adapter sequences, and paired-end reads were merged using a custom Python script (Seqprep, J. St. John, http://github.com/jstjohn/SeqPrep). Only merged reads corresponding to RNAs at least 15 nucleotides long were analyzed further. Reads were mapped to reference genomes (Homo sapiens 2009 assembly hg19, GRCh37 or S. cerevisiae April 2011 assembly sacCer3) plus the set of mature tRNA sequences from tRNAscan-SE tRNA gene predictions for each of these genomes19. Mature tRNA sequences were generated to account for post-transcriptional processing steps: predicted introns were removed, a CCA sequence was added to the 3′ ends of all tRNAs, and a G nucleotide was added to the 5′-end of histidine tRNAs. Each of these mature tRNA sequences were padded on both ends with 20 “N” bases to allow mapping of reads with additional end sequences. Reads were mapped to the reference genomes plus the non-redundant set of predicted mature tRNA sequences using Bowtie 2, returning up to 100 alignments per read with default parameters. For analyses summarizing the composition of RNA-seq reads by RNA class, multiple mapping was not allowed and only the Bowtie 2 primary alignment was used (selected arbitrarily by the program when multiple features produced equal mapping scores). |
| Organism/cell line | Saccharomyces cerevisiae, Homo sapiens (GM12878 B-cell line and GM05372 B-cell lymphoma-derived) |
| Conditions | AlkB-treated vs untreated |
| Approximate experimental time | 1-2 days |
| Starting RNA amount | 50 µg total RNA |
| tRNA expression | + |
| Base modifications | + |
| Charging status | - |
| tRNA processing and fragmentation | ++ |
| Citation | https://doi.org/10.1038/nmeth.3508 |
