|
| Status |
Public on Oct 11, 2021 |
| Title |
ONT_native_WT1 |
| Sample type |
SRA |
| |
|
| Source name |
Neural progenitor cells
|
| Organism |
Mus musculus |
| Characteristics |
genotype: Wild type
cell type: Neural progenitor cells
sequencing technology: Oxford Nanopore Technology Native RNA long-read sequencing
|
| Growth protocol |
Mouse NPCs were isolated from Mettl14 WT and cKO mouse embryonic cortices and cultured in Neurobasal medium (GIBCO BRL) containing 20 ng/ml FGF2, 20 ng/ml EGF, 5 mg/ml heparin, 2% B27 (v/v, GIBCO BRL), Glutamax (Invitrogen), Penicillin/Streptomycin (Invitrogen) on culture dishes precoated with Matrigel matrix (2%, Corning).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using TRIzol from cell pellets following the manufacturer's protocol.
For native RNA sequencing libraries were prepared by the standard kit (SQK-RNA002, Oxford Nanopore Technologies). The libraries were loaded onto R9.4.1 flowcells (FLO-MIN106D, Oxford Nanopore Technologies) and sequenced using four MinION Mk1b devices separately in parallel (MinKNOW 4.1.2, Oxford Nanopore Technologies). For direct cDNA sequencing, Each 1.5 μg of purified total RNA with 0.1 μl of RCS from direct RNA sequencing kit (SQK-RNA002) and 0.1 ng of the SIRV set 3 (Lexogen) control was prepared as a sequencing library following manufacturers instruction (SQK-DCS109, Oxford Nanopore Technologies).
ONT long-read RNA sequencing
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
MinION |
| |
|
| Description |
ONT_native_WT_flair.gtf and ONT_native_WT_stringTie.gtf
|
| Data processing |
ONT reads were aligned to Ensembl mouse genome assembly GRCm38 using minimap2 v2.17- r941. Following recommendations at https://github.com/lh3/minimap2, we used option -axsplice to allow spliced alignments and provided splice junctions extracted from the corresponding Ensembl release 95 transcriptome annotation with parameter --junc-bed. In the alignment of native RNA reads, we additionally used options -k14 -uf as recommended.
We used FLAIR v1.5.1 to identify and StringTie2 to assemble transcripts from ONT reads. We ran FLAIR with default settings on pooled reads from both WT and KO replicates and extracted condition-specific transcripts that had an estimated count of at least 1 in at least one of the 2 replicates per condition.
GRCm38/mm10
gtf files contain all identified transcripts per sample
|
| |
|
| Submission date |
Jun 10, 2021 |
| Last update date |
Oct 11, 2021 |
| Contact name |
Francisca Rojas Ringeling |
| Organization name |
Ludwig Maximilian University of Munich
|
| Department |
Biochemistry
|
| Lab |
Stefan Canzar
|
| Street address |
Feodor-Lynen-Straße 25, 81377
|
| City |
Munich |
| ZIP/Postal code |
81377 |
| Country |
Germany |
| |
|
| Platform ID |
GPL24973 |
| Series (1) |
| GSE158985 |
Partitioning RNAs by length improves transcriptome reconstruction from short-read RNA-seq data |
|
| Relations |
| BioSample |
SAMN19655098 |
| SRA |
SRX11110865 |
