|
| Status |
Public on Jan 06, 2021 |
| Title |
ERCC00048_polyA_polyI_ONT |
| Sample type |
SRA |
| |
|
| Source name |
ERCC-00048
|
| Organism |
synthetic construct |
| Characteristics |
cell line: synthetic construct
rna preparation: in vitro transcription of ERCC-00048
|
| Growth protocol |
K562 cells (ATCC, CCL-243) were maintained at 37°C and 5% CO2 in RPMI 1640 medium containing 10% FBS, 100 U/ml penicillin and 100 ug/ml streptomycin
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Oxford Nanopore Technologies direct RNA sequencing with SQK-RNA002 kit
|
| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
MinION |
| |
|
| Description |
ERCC-00048 with poly(A) and poly(I) tails addition
|
| Data processing |
Library strategy: nano-COP
Raw signal fast5 files were basecalled during sequencing with MINKNOW. RNA sequences that pass basecalling thresholds were converted into DNA by substituting U to T bases before sequence alignment. Sequences were aligned to the reference genomes using minimap2 (version 2.10-r764-dirty) with recommended parameters for Oxford Nanopore Technologies direct RNA sequencing (-ax splice -uf -k14) or cDNA sequencing (-ax splice). All analyses were performed using reads that pass the MINKNOW sequencing threshold (QC>7) and align uniquely to the genome.
The splice_df datasets were generated by first intersecting reads with constitutively spliced introns. Constitutively spliced introns with “medium stringency” included in this dataset have at least 20 reads total RNA-seq dataset that span either splice junction by at least 4 nt overlap and more than 80% of the spanning reads are spliced. For reads mapping to constitutive introns, the features of read cigar strings were extracted for the 50 nt around the intron 5’ and 3’ splice sites and the entirety of the intron. Reads were called as ‘not spliced’ if the alignment file shows no indication of splicing within the 50 nt around each splice site, mapped portions of the read (rather than deletions) represent greater than 50% of the 50 nt around each splice site, and at least 75% of the read within the intron is aligned to the reference. Reads were called as ‘spliced’ if the alignment file displays the start or end of a splicing event within the 50nt around both splice sites and the size of the aligned splicing event is within 90-110% and 100 nt of the intron size. If aligned reads that map to introns do not meet these qualifications, the splicing event is characterized as ‘undetermined’ and not used in subsequent analyses. Other information included in this file include the soft clipped length at the start of the read alignment, the distance between the read start (or aligned RNA end) and the 3’SS of the intron, and whether or not the read start (or aligned RNA end) is close to the poly(A) site of a gene (within 150 nt upstream or any distance downstream of the annotated poly(A) site of the gene)
The polyA_polyI_estimates datasets were obtained with nanopolish_polyI using default parameters
Genome_build: hg38
|
| |
|
| Submission date |
Jul 09, 2020 |
| Last update date |
Jan 07, 2021 |
| Contact name |
Karine Choquet |
| E-mail(s) |
karine.choquet@usherbrooke.ca
|
| Organization name |
Université de Sherbrooke
|
| Department |
Biochemistry and Functional Genomics
|
| Street address |
3201 rue Jean-Mignault
|
| City |
Sherbrooke |
| State/province |
Québec |
| ZIP/Postal code |
J1E 4K8 |
| Country |
Canada |
| |
|
| Platform ID |
GPL25738 |
| Series (1) |
| GSE154079 |
Revealing nascent RNA processing dynamics with nano-COP |
|
| Relations |
| BioSample |
SAMN15491756 |
| SRA |
SRX8698259 |
