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GSE132971

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Direct RNA sequencing enables single-nucleotide m6A detection in endogenous transcript isoforms

Organism: Homo sapiens
Platform: GPL24106
Samples: 4
Experiment Types:
Expression profiling by high throughput sequencing
Submitted: Jun 19 2019
Last Updated: Jan 14 2020
Status: Public on Jan 14 2020
Contact: Gene,,Yeo (UCSD)

Relations

BioProject: https://www.ncbi.nlm.nih.gov/bioproject/PRJNA549597 SRA: https://www.ncbi.nlm.nih.gov/sra?term=SRP213119

Summary

We report the direct RNA sequencing of HEK293 and a primary human mammary epithelial cell (HMEC) line using Oxford Nanopore based sequencing. Using this data, we built an algorithm to detect m6A modifications within the DRACH motif context. Evaluation of m6A sites was carried out with HEK METTL3 knockdown and HMEC ALKBH5 over expression cell lines.

Overall Design

Total RNA was isolated from HEK and HMEC cell lines using Trizol or Direct-zol, respectively. RNA was then poly-A selected using oligo-dT magnetic resin before library preparation using Oxford Nanopore’s direct RNA-sequencing kit and protocol. Sequencing output was mapped to reference genomes and resquiggled using Tombo. Tombo’s fraction modified and coverage files were subsequently combined and used as input for m6A detection.

Analysis (4 steps)

View Data Processing
Processing steps for GSE132971
  1. Oxford Nanopore's Albacore and Guppy base-callers
  2. Reads were aligned and resquiggled with Tombo v1.4
  3. Coverage and fraction modified files were obtained using Tombo's text_output commands.
  4. Bedobs was used to convert the wig files to bed files before being interescted with the coverage file using bedtools.

Supplementary Files (1)

GSE132971_RAW.tar Download
GEO Samples (4)
GSM3897645 GSM3897646 GSM3897647 GSM3897648

Dataset Citations (1)

Direct RNA sequencing enables m<sup>6</sup>A detection in endogenous transcript isoforms at base-specific resolution.
PMID 31624092 · 2020 · RNA (New York, N.Y.)
Daniel A Lorenz, Shashank Sathe, Jaclyn M Einstein, Gene W Yeo

SRA Experiments (4) and Runs (4)

Total: 4272 MB
SRX6407950 SRP213119 RNA-Seq SINGLE
GSM3897645: HEK293_WT; Homo sapiens; RNA-Seq
Sample: SRS5065406
BioProject: PRJNA549597
BioSample: SAMN12094240
Platform: OXFORD_NANOPORE
Instrument: MinION
Organism: Homo sapiens
Sample attributes
source_name: HEK293_WT
cell line: HEK293
genotype/variation: WT
Original files (1)
HEK293_WT
Runs (1)
Run Spots Bases Size (MB) Files Link
SRR9646141 1440957 1554640823 1179.59 HEK_WT_Fast5.tar.gz, SRR9646141, SRR9646141.lite SRA
SRX6407951 SRP213119 RNA-Seq SINGLE
GSM3897646: HEK293_shMETTL3; Homo sapiens; RNA-Seq
Sample: SRS5065407
BioProject: PRJNA549597
BioSample: SAMN12094239
Platform: OXFORD_NANOPORE
Instrument: MinION
Organism: Homo sapiens
Sample attributes
source_name: HEK293_shMETTL3
cell line: HEK293
genotype/variation: shMETTL3
Original files (1)
HEK293_shMETTL3
Runs (1)
Run Spots Bases Size (MB) Files Link
SRR9646142 1104708 1024791909 748.87 HEK_shMETTL3_Fast5.tar.gz, SRR9646142, SRR9646142.lite SRA
SRX6407952 SRP213119 RNA-Seq SINGLE
GSM3897647: HMEC_WT; Homo sapiens; RNA-Seq
Sample: SRS5065408
BioProject: PRJNA549597
BioSample: SAMN12094238
Platform: OXFORD_NANOPORE
Instrument: MinION
Organism: Homo sapiens
Sample attributes
source_name: HMEC_WT
cell line: Human Mammary Epithelial Cells (HMEC)
genotype/variation: WT
Original files (1)
HMEC_WT
Runs (1)
Run Spots Bases Size (MB) Files Link
SRR9646143 1814082 1686268739 1278.68 HMEC_WT_Fast5.tar.gz, SRR9646143, SRR9646143.lite SRA
SRX6407953 SRP213119 RNA-Seq SINGLE
GSM3897648: HMEC_ALKBH5; Homo sapiens; RNA-Seq
Sample: SRS5065409
BioProject: PRJNA549597
BioSample: SAMN12094237
Platform: OXFORD_NANOPORE
Instrument: MinION
Organism: Homo sapiens
Sample attributes
source_name: HMEC_ALKBH5
cell line: Human Mammary Epithelial Cells (HMEC)
genotype/variation: ALKBH5
Original files (1)
HMEC_ALKBH5
Runs (1)
Run Spots Bases Size (MB) Files Link
SRR9646144 1660731 1394864280 1064.41 HMEC_ALKBH5_Fast5.tar.gz, SRR9646144, SRR9646144.lite SRA

Linked Publications (1)

Direct RNA sequencing enables m<sup>6</sup>A detection in endogenous transcript isoforms at base-specific resolution.
RNA (New York, N.Y.) · 2020

Data Files (4)

Accession File Name Stored Type Output Type Mapping Assembly Size Download
— HEK_shMETTL3_Fast5.tar.gz RNA-Seq 748.9 MB link
— HEK_WT_Fast5.tar.gz RNA-Seq 1.2 GB link
— HMEC_ALKBH5_Fast5.tar.gz RNA-Seq 1.0 GB link
— HMEC_WT_Fast5.tar.gz RNA-Seq 1.2 GB link