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| Status |
Public on Jul 11, 2024 |
| Title |
miCLIP_mESC_NSUN5_Eluent_rep1 |
| Sample type |
SRA |
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| Source name |
cell line
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| Organism |
Mus musculus |
| Characteristics |
tissue: cell line
cell line: mESC
cell type: Mouse embryonic stem cells
genotype: NSUN5 C308A overexpression
treatment: Cells were lysed and followed by partial RNA digestion with RNase I. Then, FLAG immunoprecipitation was performed using anti-FLAG magnetic beads. The protein-RNA complex was eluted and a second biotin immunoprecipitation was conducted with streptavidin beads. The RNA was purified by RNA Isolater and subjected to library construction.
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| Growth protocol |
The mESC cell line was cultured in a complete ESC culture medium, comprising Dulbecco's modified Eagle's medium (DMEM) supplemented with 15% heat-inactivated fetal bovine serum (FBS), 0.1 mM 2-mercaptoethanol, 2 mM L-glutamine, 0.1 mM non-essential amino acids, 1000 U/mL recombinant leukemia inhibitory factor (LIF), and 100 U/mL penicillin/streptomycin. For differentiation assays, mESC cells were cultured without LIF for six days. HeLa and HEK 293T cell lines were cultured in DMEM supplemented with 10% FBS and 100 U/mL penicillin/streptomycin.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extraction from the cell lines was accomplished using the RNA isolater total RNA extraction reagent (Vazyme, R401-01). Prior to size selection, RNA underwent DNase I treatment at 37 ℃ for 15 min and subsequently was purified using RNA isolater extraction and ethanol precipitation (Vazyme, EN401-01). The small RNA fraction, characterized by a size of less than 200nt, was enriched from the total RNA utilizing the RNA Clean & Concentrator-25 Kit (Zymo, R1018). For mRNA purification, two successive rounds of poly(A)+ selection were performed employing oligo(dT)25 dynabeads (Life Technologies, 61005). Additionally, chromatin-associated RNA was isolated following subcellular fractionation assay, DNase I treatment, and rRNA depletion (Vazyme, N406-01). Modification-free control RNAs were prepared from HeLa or mESC mRNA, adhering to a previously published protocol (PMID: 34594034, 36593412).
The RNA samples were initially dephosphorylated using T4 polynucleotide kinase (Vazyme, N102-01) at 37℃ for 1 hour, followed by ethanol precipitation. Subsequently, they were ligated to the 3' RNA linker using T4 RNA ligase 2, truncated at 25°C for 2 hours, followed by an overnight incubation at 16°C (Beyotime, R0635L). To remove excess adaptors, the samples underwent treatment with 5´ Deadenylase (NEB, M0331S), RecJf (NEB, M0264S), and Proteinase K (NEB, P8102). RNA samples intended for group comparisons were pooled and then purified through Tris-phenol extraction followed by ethanol precipitation. The barcoded and pooled samples were labeled by 5-(2-azidoethyl)-1,3-indandione (AI) and subsequently subjected to a click reaction. The m5C-modified RNA was subjected to RNA pull-down to be enriched, followed by reverse transcription using the HiScript III RT SuperMix (Vazyme, R323-01). The excess reverse transcription primer was digested with exonuclease I (NEB, M0293S). The cDNA was purified by silane beads and then performed to 5′ adapter ligation on beads with T4 RNA ligase 1, high concentration (NEB, M0437M). cDNA was purified using silane beads (Invitrogen, 37002D), and subsequently, the purified cDNA was amplified using the KAPA HiFi HotStart ReadyMix (Roche, KK2602). Finally, PCR products were purified using 1.8X VAHTS DNA Clean Beads (Vazyme, N411-02) and 7% TBE gel.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
For batch paired-end reads of m5C-seq libraries, a sample-barcoded-sequence (Pos 1-6) and random-4-base (pos7-10) in Read1 and a 10nt unique molecular identifier (UMI) was included in the adaptor that ligates to the 3’end of cDNA so it locates in Read2 (Pos1-10). Firstly, m5C-seq batch libraries were demultiplexed into individual samples, using fastq-multx（with -m 1 parameters）that distributes reads into a sample FASTQ file(Read1.fq file and Read2.fq file) according to the sample-barcoded-sequence in Read1 (Pos 1-6). For every individual sample, only Read2 data with UMI was used for subsequent analyses. Illumina sequencing reads were first treated with trim_galore(version 0.6.4) for adapter removal and quality trimming. Work commands are as follows: trim_galore --fastqc --phred33 --length 30. We then used Seqkit (version 0.10.0) to deduplicate based on 10 bp UMI at the 5′ end of reads R2, key process parameters are as follows: seqkit rmdup -s. Finally, Fastp (version 0.23.2) was used to remove the 10 bp UMI in the deduplication read. 10 bases at the 3′ end of inserted sequences were also removed, using the key parameters: fastp -f 10 -t 10 -l 20.
Processed reads were mapped to reference genome (human GRCh19 reference / mouse mm10 reference) using HISAT2-3N (version 2.2.1-3n-0.0.3). The key parameters are as follows: hisat3n --base-change C,T --directional-mapping --repeat -p 20 -k 1. Considering a creditable m5C signal, the alignments are further filtering using python house-scripts, the alignments hosts >=3 C-to-T mismatches or >= 3 other type mismatches or the length of alignments < 20nt are filtered. Next, we parse BAM file using pysamstats (version 1.1.2), with commands as follow: pysamstats -D 100000 -S "nofilter" -t variation.
To identify the methyltransferase-dependent m5C sites, we calculated the C-to-T ratios for the m5C-TACseq-identified m5C sites in WT and all knockout/knockdown samples. Then, the fold change and difference of C-to-T ratio between KO/KD samples and WT/Control samples were calculated. For the identification of enzyme-dependent sites by comparing the KO and WT samples, the fold change in C-to-T ratio (KO sample/WT sample) was required to be less than 60% in two replicates, and the difference in C-to-T ratio (WT sample - KO sample) was required to be > 1.5%. Considering the presence of residual enzymes upon knockdown, to identify enzyme-dependent sites by comparing the KD and Control samples, the fold change in C-to-T ratio (KD sample/Control sample) was required to be less than 60% in at least one replicate.
The analysis of CLIP-seq data was performed using CLIP Tool Kit (CTK, v1.1.3). The processed reads were mapped to the mouse genome (Ensembl annotation of mm10) using hisat2 v2.2.1 with default settings.
Assembly: hg19(UCSC)/mm10(UCSC)/tRNA-referenece(tRNAdb)
Supplementary files format and content: tab-delimited text file includes every C base coverage and C-to-T signal
Supplementary files format and content: excel file,in xls file format
Library strategy: m5C-seq
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| Submission date |
Jun 03, 2024 |
| Last update date |
Jul 11, 2024 |
| Contact name |
Xiaoyu Li |
| E-mail(s) |
xiaoyu_li@zju.edu.cn
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| Organization name |
Zhejiang University
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| Department |
SCHOOL OF BASIC MEDICAL SCIENCES
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| Lab |
Li Lab
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| Street address |
866 Yuhangtang Road
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| City |
Hangzhou |
| State/province |
Zhejiang |
| ZIP/Postal code |
310058 |
| Country |
China |
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| Platform ID |
GPL24247 |
| Series (1) |
| GSE242724 |
Base-resolution m5C profiling across the mammalian transcriptome by bisulfite-free enzyme-assisted chemical labeling approach |
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| Relations |
| BioSample |
SAMN41656085 |
| SRA |
SRX24781297 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8302351_NSUN5-Eluent-rep1.F.bw |
394.0 Kb |
(ftp)(http) |
BW |
| GSM8302351_NSUN5-Eluent-rep1.R.bw |
357.1 Kb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
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