| Full name | demethylation-assisted multiple methylation sequencing |
| Summary | Method allows us to monitor the modification level based on the mutation rate for m1A, m3C, m1G and m22G sites using engineered AlkB |
| Key findings | Loss of ALKBH7 leads to increased mt-tRNAIle m22G and mt-tRNALeu1 m1A on mt-dsRNA, disrupts mitochondrial RNA processing, reduces mt-tRNA levels and translation, and compromises mitochondrial activity |
| Sequencing strategy | RNA-Seq |
| Raw data | https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE148202 |
| Experimental protocol | Cellular total RNA was isolated with TRIzol reagent (Invitrogen) following the manufacturer’s protocol by isopropanol precipitation. The small RNA fraction (size < 200 nt) was further extracted from the purified total RNA using the mirVana miRNA Isolation Kit (AM1560, Invitrogen). A 300- to 500-ng quantity of human cellular small RNA (size < 200 nt) was fragmented into 40- to 50-nt lengths using RNA Fragmentation Reagent (AM8740, Invitrogen) following the manufacturer’s protocol and purified by Oligo Clean & Concentrator (Zymo Research). About 50 ng was saved as input and the remainder was subjected to an optimized demethylation treatment (30 mM MES buffer pH 5.5, 100 μM (NH4)2Fe(SO4)2·6H2O 300 μM α-ketoglutarate, 2 mM l-ascorbic acid, 150 mM NaCl, 2 mM MgCl2 and 40 μg ml−1 BSA) with 2.5 μl SUPERase•In RNase Inhibitor and 1.0 μl of engineered AlkB (D135S (ref. 25), 10 mg ml−1) added to the 50-μl reaction mixture, followed by incubation at 25 °C for 2 h and then purification using Oligo Clean & Concentrator. 3′-end repair was conducted for the input and demethylase-treated RNA using T4 Polynucleotide Kinase (EK0032, Thermo Fisher Scientific). RNA was mixed with 5 μl of 10× T4 Polynucleotide Kinase Reaction Buffer (B0201S, NEB) and 5 μl of T4 PNK, diluted to a final volume of 50 μl, and incubated at 37 °C for 1 h followed by Oligo Clean & Concentrator purification. To perform 3′-adapter ligation, repaired RNA fragments were incubated with 1.0 μl of 30 μM RNA 3′-linker (5′rApp-NNNNNAGATCGGAAGAGCGTCGTG-3SpC3) at 70 °C for 2 min and immediately placed on ice. A 2.5-μl volume of 10× T4 RNA Ligase Reaction Buffer (NEB), 7.5 μl of 50% PEG8000, 1 μl of SUPERase•In RNase Inhibitor and 2 μl of T4 RNA ligase 2 truncated KQ (NEB) were added to the RNA–adapter mixture. The reaction was diluted to a final volume of 25 μl and incubated at 25 °C for 2 h followed by 16 °C for 10 h. The reaction was further diluted to 47 μl with nuclease-free water, and the excessive adapters were removed with 2 μl of 5′-deadenylase (NEB) at 30 °C for 1 h followed by adding 1.0 μl of RecJf (NEB) for ssDNA digestion at 37 °C for 1 h. The 3′-end-ligated RNA was extracted by RNA Clean & Concentrator (Zymo Research).The purified RNA was incubated with 1.0 μl of 2.0 μM RT primer (5′-ACACGACGCTCTTCCGATCT-3′) at 65 °C for 2 min and immediately moved to ice. A 2-μl volume of 10× AMV Reverse Transcriptase Reaction Buffer (NEB), 2 μl of 10 mM dNTP Solution Mix (NEB), 1 μl of RNaseOUT Recombinant Ribonuclease Inhibitor (Thermo Fisher Scientific) and 2 μl of HIV Recombinant Reverse Transcriptase (Worthington) were added to the RNA–primer mixture. The reaction was diluted to 20 μl and incubated at 37 °C for 90 min. A 1.0-μl volume of RNase H (NEB, M0297L) and 2.5 μl 10× RNase H Reaction Buffer were then added and the reaction was incubated at 37 °C for 20 min. The cDNAs were purified and eluted in 7 μl by Oligo Clean & Concentrator (Zymo Research). A mixture of cDNA and 1.0 μl of 50 μM cDNA 3′-linker (5′Phos-NNNNNAGATCGGAAGAGCACACGTCTG-3SpC3) was denatured at 75 °C for 2 min and then left on ice. A 3-μl volume of 10× T4 RNA Ligase Reaction Buffer (NEB), 15 μl of 50% PEG8000, 3 μl of 10 mM ATP and 1.0 μl of T4 RNA ligase 1 (high concentration, NEB) were added to the cDNA–adapter mixture, and the reaction was incubated at 25 °C for 12 h. The libraries were amplified using universal and indexed primers from NEBNext Multiplex Oligos for Illumina (NEB). All libraries were sequenced on an Illumina NovaSeq 6000 with a single-end read length of 100 bp. |
| Analysis protocol | The sequencing data were all trimmed with the cutadapt tool to remove adapters and low-quality reads (length shorter than 18 bp). PCR duplicates were removed with the BBMap tool (https://sourceforge.net/projects/bbmap/), random barcodes at reads end were trimmed, and low-quality reads were removed using the cutadapt tool. The remaining reads were aligned to the human genome (hg19) using Tophat2 (version 2.1.1) and bowtie2 (version 2.3.5.1) allowing a maximum of three mismatches. The generated .bam files were split into positive and negative strands and sorted using Samtools. Sequence variants were identified by measuring the base composition at each position using fine-tuned bam-readcount (https://github.com/genome/bam-readcount). The generated bam-readcount output results were parsed and analysed to calculate the misincorporation ratio at each methylated site (m1A, m1G, m22G and m3C) in mt-tRNA or mt-dsRNA, followed by confirmation using direct visualization through IGV software (https://software.broadinstitute.org/software/igv/). |
| Organism/cell line | Homo sapiens (HepG2, HeLa) |
| Conditions | cellular small RNA samples from HepG2 cells; mitochondrial tRNA samples to observe methylation fraction change in vivo, including siALKBH7 vs. siControl, shTRMT1 vs. shControl and o/e ALKBH7 vs. o/e Control in HepG2 and HeLa cell lines; mitochondrial dsRNA samples to observe methylation fraction change in vivo, including siALKBH7 vs. siControl, ALKBH7 wild-type vs. ALKBH7 knockout, shTRMT1 vs. shControl and o/e ALKBH7 vs. o/e Control in HepG2 and HeLa cell lines; HepG2 EU-labeled RNA samples to observe mt-mRNA and junction level change in vivo, including 10-min EU-labeled RNA from siALKBH7 vs. siControl, 10-min EU-labeled RNA from wild-type vs. ALKBH7 KO HepG2 cells, 20-min EU-labeled RNA from wild-type HepG2 cells and 30-min EU-labeled RNA from wild-type HepG2 cells |
| Approximate experimental time | 3-4 days |
| Starting RNA amount | 50 ng small RNA |
| tRNA expression | ++ |
| Base modifications | + |
| Charging status | - |
| tRNA processing and fragmentation | - |
| Citation | https://doi.org/10.1038/s41556-021-00709-7 |
