Dimethyl-sulfate chemical probing and mutational profiling coupled with demethylase treatment in transcriptome-wide tRNA sequencing
| Full name | dimethyl-sulfate chemical probing and mutational profiling coupled with demethylase treatment in transcriptome-wide tRNA sequencing |
| Sequencing strategy | RNA-seq |
| Raw data | https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE262888 |
| Software/data repository | https://github.com/ckatanski/MSR-seq |
| Experimental protocol | In vivo DMS treatment of HEK 293T cells. To a 10-cm dish of HEK 293T cells cultured in 15 ml media, 300 µL or 750 µL of DMS were added to reach a final concentration of 2% or 5%, respectively. After the addition of DMS, cells were incubated at 37 °C for 5 min. The media was then aspirated off, and the cells were washed with PBS twice and harvested for total RNA extraction with TRIzol (Thermo Fisher, 15596026) following the manufacturer’s protocol. In vitro DMS treatment of HEK 293T total RNA. Total RNA was extracted from 80 to 90% confluent HEK 293T cells with TRIzol (Thermo Fisher, 15596026) following the manufacturer’s protocol. 4 µg of HEK 293T total RNA in 10 µL H2O was combined with 10 µL of the 2× refolding buffer (200 mM NaCl, 12 mM MgCl2, 20 mM Tris, pH 8.0) and incubated at 30 °C for 30 min. DMS was added to each sample to reach a final concentration of 5% at room temperature for 5 min. DMS treatment was stopped by adding an equal volume of 2× DMS quench buffer (60% β-mercaptoethanol, 0.6 M NaOAc, pH 5.5). DMS-treated total RNA samples were precipitated with ethanol. Approximately 1 µg of in vivo or in vitro DMS treated-total RNA samples were used to build tRNA sequencing libraries, following a previously published MSR-seq protocol24. Briefly, RNAs were first deacylated by incubating at 37 °C in a 33 mM sodium tetraborate buffer for 30 min. Next, 5 µL of a PNK reaction stock (4 U/µL NEB T4 PNK, 40 mM MgCl2, 200 mM Tris-HCl, pH 6.8) was added to repair the 3’ end, and the sample was incubated at 37 °C for 20 min and then incubated at 65 °C for 10 min to inactivate the PNK enzyme. Following 3’ end repair, 30 µL of an RNA ligation reaction mix was added to the sample while still in the 3’ end repair mix (final concentration: 15% PEG 8000, 1× T4 RNA ligase I buffer, 50 µM ATP, 5% DMSO, 1 mM hexaamide cobalt (III) chloride, and 1 U/µL T4 RNA ligase I) and incubated overnight at 16 °C. This mix also included the barcoded RNA ligation linker/RT primer oligo at a 1.2:1 molar ratio to the input RNA. After ligating the RNA overnight, the sample was bound to streptavidin-coated MyOne C1 dynabeads (ThermoFisher) at room temperature on rotation for 15 min to facilitate the library construction process by minimizing sample loss and enabling rapid washes and buffer exchanges between reactions. “+DM” samples were treated according to the established demethylase protocol below. “+DM” and “−DM” samples were then dephosphorylated using a CIP reaction mix (0.2 U/µL CIP, 10 mM MgCl2, 0.5 mM ZnCl2, 20 mM HEPES, pH 7.5) for 30 min at 37 °C to liberate the 3’ end of the RT primer. Next, samples were resuspended in 25 µL of 1x SuperScript IV VILO mix and incubated at 55 °C for 10 min and then at 37 °C overnight. Next, samples were treated with an RNase H reaction mix (0.4 U/µL NEB RNaseH, 1× RNase H buffer) for 15 min at 37 °C. Subsequently, samples were treated with a solution of 50 mM sodium periodate in 15 mM sodium acetate, pH 5.0, for 30 min at room temperature to oxidize any non-extended RT primer. Following this, an overnight, room temperature cDNA ligation step was performed to enable PCR amplification of the cDNA (50 µL reaction; final concentrations: 2 U/µL T4 RNA ligase I, 25% PEG 8000, 7.5% DMSO, 50 µM ATP, 1 mM hexaamine cobalt (III) chloride, 2 mM barcoded cDNA ligation oligo, 1× T4 RNA ligase I buffer). Finally, the libraries were amplified by PCR with Illumina primers. Sequencing was conducted using Illumina NovaX 6000, 100-bp paired-end. |
| Analysis protocol | Raw sequences reads were demultiplexed using Je to detect encoded barcode reads. Following this, reads were aligned to curated tRNA reference genome. The aligned reads were then used to determine RNA sequence abundance using custom python script. RNA modifications were detected based on aligned reads using samtools sort ( feature sort the reads in a bam file format. Then IGVtools count count feature was utilized to output a wig files using the following parameters: -z 5 -w 1 -e 250 —bases. The resulting wig files were processed using a custom python script to identify nucleotide mutations as well as coverage of aligned reads. Assembly: hg38 Supplementary files format and content: tsv files with gene name and abundance per sample Supplementary files format and content: .sam.tsv files with mutation, deletion, coverage for each gene position per sample |
| Organism/cell line | Homo sapiens (HEK293T) |
| Conditions | in vitro vs in vivo; unstressed vs oxidative stress |
| Approximate experimental time | 3-4 days |
| Starting RNA amount | no data about total amount of material used |
| tRNA expression | ++ |
| Base modifications | + |
| Charging status | + |
| Citation | https://doi.org/10.1038/s41467-025-59435-5 |