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| Status |
Public on Apr 24, 2024 |
| Title |
HeLa_small_RNA_NaBH4_PSII.rep2 |
| Sample type |
SRA |
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| Source name |
HeLa
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| Organism |
Homo sapiens |
| Characteristics |
cell line: HeLa
genotype: Wide type
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| Growth protocol |
HeLa cells (American Type Culture Collection) were cultured in DMEM medium (Gibco™, catalog no. 11995065) supplemented with 10% (v/v) fetal bovine serum (Gibco™ #16000044), 100 U/mL of penicillin and 100 μg/mL streptomycin (Gibco™ #15140122) at 37 °C with 5% CO2.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA from cultured cells was extracted with TRIzol™ Reagent (Invitrogen™, Catalog no. 15596026) following manufacturer’s instructions. Small RNA fraction was enriched from total RNA using RNA Clean & Concentrator-25 (Zymo, Catalog no. R1017).
500 ng of HepG2 small RNA (<200 nt) was mixed with 0.5 ng of 43-mer m1A RNA oligo and 1 ng of 45-mer m1A RNA oligo. 1x T4 PNK buffer (NEB), 0.4 U/μL SUPERase•In (Invitrogen) and 0.8 U/μL T4 PNK (NEB M0201) were added and mixture was incubated at 37 °C for 45 min. Next, RNA was purified with RNA Clean & Concentrator-5 (Zymo Research) and eluted with 21 μL water. RNA concentration was measured with Qubit (Invitrogen). RNA volume was then adjusted to 36 μL and split to set up four 3’ RNA ligation reactions. ~100 ng RNA of RNA in 9 μL water was mixed with 1 μL of 11.25 μM 3’ RNA adaptor and incubated at 70 °C for 2 min then kept on ice for 2 min. Next, 2 μL water, 2.5 μL of 10X T4 RNA Ligase Reaction Buffer (NEB B0216S), 7.5 μL of 50% PEG8000 (NEB B1004S), 1 μL of SUPERase•In and 2 μL of T4 RNA ligase 2 truncated KQ (NEB 0373) were added and the mixture was incubated ar 25 °C for 1 h followed by incubation at 16 °C for 16 hr. Next day, to each ligation mixture, 1 μL of 5’ deadenylase (NEB M0331) was added and incubated at 30 °C for 1h. Adaptor excess was digested by adding to each ligation mixture 1 μL of RecJf (NEB M0264) and incubating at 37°C for 1h. After adaptor digestion, all four ligation mixtures were combined and purified with RNA Clean & Concentrator-5. To further remove undigested 3’ adaptor, EtOH volume was adjusted. Briefly, ligation reactions mixture volume was adjusted to 400 μL with water. Then, 400 μL RNA Binding Buffer (Zymo) and 320 μL of 100% EtOH were added to the sample and then loaded onto the column. Further steps were performed according to manufacturer’s instructions. Purified 3’ RNA ligated sample was then divided into three parts: (i) to treat with NaBH4, (ii) to treat with pNTP, (iii) to treat with Tris-HCl pH 8.8. Each part was further subdivided into three parts as technical replicates. Treatment was performed as follows: (i) Store 3’ ligated RNA to treat with NaBH4 at -80 °C until ready to use. (ii) pNTP treatment was performed as described previously (REF). Briefly, to 3’ ligated RNA in 25 μL water, add 25 μL of 2x reaction mixture (2x stock: 50 mM pNTP (Thermo Scientific Chemicals H59240.14), 10 mM THP (Sigma-Aldrich 777854) in 10 %(v/v) DMF (Sigma-Aldrich 270547), pH 6). Incubate at 50 °C for 16 h. (iii) To 3’ ligated RNA in 40.5 μL water, add 4.5 μL of 1.5 M Tris-HCl pH 8.8 and incubate at 95 °C in a lid-heated to 110 °C thermoblock for 16 h. Next, add 5 μL of 1 M NaHCO3 pH 9.2 and incubate for additional 10 min at 95 °C. Put on ice immediately and add 10 μL of 3M NaOAc pH 5.5 buffer to neutralize the sample and prevent from further degradation. Purify pNTP- and Tris-treated samples with RNA Clean & Concentrator-5. Further steps were performed according to manufacturer’s instructions and refer the method session in the manuscript.
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| Library strategy |
ncRNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
Illumina MiSeq |
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| Data processing |
To analyze the small RNA sequencing data from HeLa cells, we initiated the process by aligning the modified reads to the hg19 reference genome assembly. Raw reverse (R1) reads with depleted PCR duplicates and removed adaptors were mapped to the rRNA (NR_003285.3.fa, NR_003286.4.fa and NR_003287.4.fa) download from NCBI Nucleotide data, and tRNA (hg19-mature-tRNAs.fa) genomes downloaded from the GtRNAdb database (http://gtrnadb.ucsc.edu/Hsapi19/Hsapi19-seq.html).
This alignment was performed using bwa mem under its default parameters. Following this, the mapped SAM files were converted to BAM format and sorted using Samtools sort (v1.9). To refine our data further, we filtered the sorted BAM files using Samtools view (-q 10) to obtain uniquely mapped reads. For mutation identification, we employed rnaseqmut utilizing the parameters "-t -s -3 -m 2" to ensure sensitive detection.
Our analysis incorporated several stringent cutoffs to filter the mutation list for downstream analysis: 1) we selected mutations with a reference Adenine (A) and a read coverage of at least 50 and a mutation ratio of 5% or greater in both SSIV and PSII libraries, coupled with a p value of less than 0.05. 2) For two control libraries, we focused on adenine sites with a read coverage of at least 50 and a mutation ratio of less than 5%.
Additionally, we required that mutation ratios in both NaBH4 SSIV and PSII libraries be at least 2-fold higher than those in two control libraries.
Assembly: mm10
Supplementary files format and content: txt, mutation list on mature tRNA
Supplementary files format and content: txt, mutation list on rRNA
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| Submission date |
Jan 19, 2024 |
| Last update date |
Apr 24, 2024 |
| Contact name |
Ruitu Lyu |
| E-mail(s) |
lvruitu@uchicago.edu
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| Organization name |
The University of Chicago
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| Department |
Chemistry
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| Lab |
Chuan He
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| Street address |
929 E 57th Street
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| City |
Chicago |
| State/province |
IL |
| ZIP/Postal code |
60637 |
| Country |
USA |
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| Platform ID |
GPL15520 |
| Series (1) |
| GSE253657 |
Chemical manipulation of m1A mediates its detection in human tRNA |
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| Relations |
| BioSample |
SAMN39500196 |
| SRA |
SRX23309251 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8024682_HeLa_small_RNA_NaBH4_PSII.rep2.mature_tRNA.mutation.txt.gz |
74.0 Kb |
(ftp)(http) |
TXT |
| GSM8024682_HeLa_small_RNA_NaBH4_PSII.rep2.rRNA.mutation.txt.gz |
162.1 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
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