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| Status |
Public on Jul 10, 2024 |
| Title |
PUS3-/- mock treated biol rep 3 |
| Sample type |
SRA |
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| Source name |
mock
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| Organism |
Homo sapiens |
| Characteristics |
tissue: mock
cell line: HEK293TRex FlpIn
cell type: Human embryonic kidney
genotype: PUS3-/-
treatment: total RNA extraction with TRIzol, polyA enrichment, mock labelling of pseudouridines during library preparation
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| Treatment protocol |
no treatment
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| Growth protocol |
HEK293TRex FlpIn cells were grown in Dulbecco's Modified Eagle's Medium DMEM (D5671, Sigma) supplemented with 10% FBS (G3031P-500, Lucerna-Chem), 100 units penicillin, 100 µg streptomycin (P4333, Sigma), 2 mM Ala-Gln (G8541, Sigma) in a humidified incubator at 37 °C with 5% CO2.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
total RNA was extracted using 1ml TRIzol per 10cm dish following the manufacturers protocol. 200µg total RNA per replicate was used for the construction of sequencing libraries.
Pseudo-seq libraries were prepared as described before (Carlile et al., 2015) with some adaptations. Poly-A RNA enrichment was performed with Poly(A)Purist™ MAG Kit (AM1922, Thermofisher) using 200 µg total RNA input. RNA was fragmented in 10 mM ZnCl2 for 55 s at 94 °C and quenched with 20 mM EDTA followed by ethanol precipitation. RNA was then either CMC treated (0.4 M, +CMC) or mock treated (-CMC) in BEU buffer for 45 min at 40 °C and 1000 rpm. After ethanol precipitation, unspecific CMC labelling was removed by incubating the RNA in sodium carbonate buffer for 2 h at 50 °C and 1000 rpm followed by ethanol precipitation. RNA ends were repaired with T4 PNK (M0201L, NEB) for 2 h at 37 °C, precipitated and size selected (120-140 nt) by Urea-PAGE. A 3′ adenylated adapter (5′-AppGATATCGTCAAGATCGGAAGAGCACACGTCTGAA-ddC-3′) was then ligated to the RNA fragments using T4 RNA ligase (M0373L, NEB) for 4 h at 22 °C followed by ethanol precipitation. The reverse transcription was performed with AMV RT (M5108, Promega) and RT primer: 5′-pRNAGATCGGAAGAGCGTCGTGTAGGGAAAGAG-iSp18-GTGACTGGAGTTCAGACGTGTGCTC-3′. Truncated cDNAs (110-180 nt) were size selected using Urea-PAGE. Gel purified cDNA was circularized using CircLigase ssDNA ligase II (CL9025K, Epicentre) and PCR amplified with forward primer (5′-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCT*C-3′) and barcode reverse primer (NEBNext® Multiplex Oligos for Illumina®, Index Primers Set 1-4, NEB E7335S, E7500S, E7710S, E7730S). PCR products were PAGE purified and sequenced on an Illumina NovaSeq 6000 using an SP or S2 flow cell in SR75 mode to yield a total of 130 million reads per library.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
Flp-In™ T-REx™ 293
processed data file contains information from all treated and untreated replicates of PUS3-/-, columns contain headers
PUS3plusandminus.txt.gz
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| Data processing |
First, technical replicates were concatenated to one fastq file. Reads were adapter clipped and random nucleotides were trimmed using Cutadapt (DOI: https://doi.org/10.14806/ej.17.1.200).
Reads were then mapped to the transcriptome (MANE GRCh38 v1.0 ensemble rna) after mitochondrial transcripts from the human cDNA collection (Homo_sapiens.GRCh38.cdna.all.fa) were included using bowtie version 2.5.0.
The analysis was performed based on calculating the ratio of 5' read ends (earlier RT stops) over the overall coverage at each transcript position as reported before (Finet et al. 2022).
Resulting SAM files were sorted and indexed using samtools version 1.15.1. Afterwards, the 5' coverage as well as the overall coverage at each transcript position was calculated using bedtools version 2.30.0 and bedops 2.4.41. Both files of all replicates (CMC and mock treated) were combined in one table.
GNU Awk 4.0.2 was used to calculate the average coverage and the average 5' coverage/coverage. The Ψ score was calculated by dividing the average 5' coverage/coverage of one condition by the average 5' coverage/coverage of another.
Assembly: MANE.GRCh38.v1.0.ensembl_rna.fna.gz
Supplementary files format and content: tab-delimited text files including transcript id, transcript position, coverage, 5' coverage, average coverage, 5' coverage/coverage, average 5'coverage/coverage, the Ψ score and nucleotide at the specific transcript position of all replicates of either WT, PUS3-/- or PUS1-/-
Library strategy: Pseudo-Seq
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| Submission date |
Feb 07, 2024 |
| Last update date |
Jul 10, 2024 |
| Contact name |
Leon Kleemann |
| E-mail(s) |
leon.kleemann@epfl.ch
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| Organization name |
EPFL
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| Street address |
Station 19
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| City |
Lausanne |
| ZIP/Postal code |
1015 |
| Country |
Switzerland |
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| Platform ID |
GPL24676 |
| Series (1) |
| GSE255287 |
The molecular basis of tRNA selectivity by human pseudouridine synthase 3 (PUS3) |
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| Relations |
| BioSample |
SAMN39860085 |
| SRA |
SRX23567243 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
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