|
| Status |
Public on Aug 08, 2023 |
| Title |
O1_liver_LongRead_RNA |
| Sample type |
SRA |
| |
|
| Source name |
liver
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6JN
Sex: male
age: 12 weeks
tissue: liver
|
| Treatment protocol |
Old as treated group
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA integrity was assessed using QubitTM RNA IQ assay kit (ThermoFisher, Q33221) before library preparation. Libraries were constructed using 25-50 µg of total RNA using the direct RNA sequencing kit (Oxford Nanopore Technologies) as previously described with modifications (Ibrahim et al. 2021, PMID: 34428294). After selection of poly(A) RNAs using Oligo d(T)25 Magnetic Beads (New England Biolabs), 15 pmoles of REL5 adapter (/5Bio/rArArUrGrArUrArCrGrGrCrGrArCrCrArCrCrGrArGrArUrCrUrArCrArCrUrCrUrUrUrCrCrCrUrArCrArCrGrArCrGrCrUrCrUrUrCrCrGrArUrCrU) was ligated to the 5′ ends of poly(A)-purified RNAs using T4 RNA ligase 1 (New England Biolabs) for 3 hours at 37°C.
REL-ligated poly(A) RNA (750 ng) was used for library preparation according to manufacturer’s protocol (Oxford Nanopore Technologies). Final libraries were quantified using Qubit 1X dsDNA High Sensitivity (HS) assay kit (Thermo Fisher) and sequenced on a MinION device using R9.4.1 flow cells (Oxford Nanopore Technologies).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
MinION |
| |
|
| Data processing |
Nanopore direct RNA sequencing data were basecalled using Guppy (v6.1.2).
Reads were subsequently mapped to mouse genome mm10 using minimap2 (version 2.24) with parameters -a -x splice -k 12 -u b --secondary=no. Basecalled reads were also separately aligned against the mouse transcriptome (Ensembl version 92) using -a -x map-ont -k 12 -u f --secondary=no.
FLAIR (v1.7.0) was used to identify and quantify novel transcripts.
DESeq2 and DRIMSeq were used for differential expression and differential isoform usage calculations respectively. Poly(A) tail lengths were extracted from sequenced reads using the nanopolish polya package.
To identify transcripts with systematic whole-molecule or poly(A) length changes across experimental conditions, we employed in-house scripts that use linear mixed models to compare replicates using the library as a random effect (https://github.com/maragkakislab/nanoplen).
Assembly: mm10
Supplementary files format and content: Count txt files generated using FLAIR/v1.7.0
|
| |
|
| Submission date |
Dec 16, 2022 |
| Last update date |
Aug 08, 2023 |
| Contact name |
Supriyo De |
| Organization name |
NIA-IRP, NIH
|
| Department |
Laboratory of Genetics and Genomics
|
| Lab |
Computational Biology & Genomics Core
|
| Street address |
251 Bayview Blvd
|
| City |
Baltimore |
| State/province |
Maryland |
| ZIP/Postal code |
21224 |
| Country |
USA |
| |
|
| Platform ID |
GPL24973 |
| Series (2) |
| GSE221122 |
Gene body DNA hydroxymethylation restricts magnitude of transcriptional changes during aging [direct RNA-seq] |
| GSE221124 |
Gene body DNA hydroxymethylation restricts magnitude of transcriptional changes during aging. |
|
| Relations |
| BioSample |
SAMN32270500 |
| SRA |
SRX18724750 |
