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| Status |
Public on Aug 02, 2023 |
| Title |
LR UM Case18 |
| Sample type |
SRA |
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| Source name |
eye
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| Organism |
Homo sapiens |
| Characteristics |
tissue: eye
cell type: uveal melanoma cell
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| Extracted molecule |
total RNA |
| Extraction protocol |
For short reads sequencing, total RNA was isolated using Trizol Reagent (Invitrogen Life Technologies), then the concentration, quality and integrity of RNA were determined by NanoDrop spectrophotometer (Thermo Scientific). Three micrograms of RNA were used as input material for the RNA sample preparations.
For long reads sequencing, total RNA was isolated using the Trizol Reagent (Invitrogen Life Technologies), and the concentration, quality and integrity were determined by NanoDrop spectrophotometer (Thermo Scientific).
For short reads sequencing, sequencing libraries were generated according to the following steps. mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in an Illumina proprietary fragmentation buffer. First strand cDNA was synthesized using random oligonucleotides and Super Script II. Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities and the enzymes were removed. After adenylation of the 3′ ends of the DNA fragments, Illumina PE adapter oligonucleotides were ligated to prepare for hybridization. To select cDNA fragments of the preferred 400-500 bp in length, the library fragments were purified using the AMPure XP system (Beckman Coulter, Beverly, CA, USA). DNA fragments with ligated adaptor molecules on both ends were selectively enriched using Illumina PCR Primer Cocktail in PCR reaction. Products were purified (AMPure XP system) and quantified using the Agilent high sensitivity DNA assay on a Bioanalyzer 2100 system (Agilent).
For long reads sequencing, A total of 1ug RNA was prepared for cDNA libraries using cDNA-PCR Sequencing Kit (SQK-PCS109) according to the instructions of Nanopore Technologies (ONT). Defined PCR adaptors were directly added to both ends of the first-strand cDNA by reverse transcriptase. After 14-cycle of PCR by LongAmp Tag (Martin, #21), the PCR products were subjected to ONT adaptor ligation using T4 DNA ligase (Martin, #21). Agencourt XP beads was used for DNA purification according to ONT protocol. The final cDNA libraries were added to FLO-MIN109 flow-cells and were sequenced on PromethION platform.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
PromethION |
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| Data processing |
Short reads were aligned to the reference human genome, GRCh38 using STAR (2.7.0d). All annotated and unannotated isoforms were merged to generate a combined isoform annotation file in “GTF” format.
With the cufflinks program and corresponding short reads data we estimated the expression level for each isoform based on the combined annotation file.
We used two different pipelines, including FLAIR and pinfish, to identify isoforms from long reads. For the FLAIR pipeline, “flair align” first aligns long reads to the reference human genome GRCh38 genome using minimap2, then “flair correct” corrects misaligned splice sites using GENCODE v24 annotations and corresponding short-read splice junctions, and finally “flair collapse” defines high-confidence isoforms from corrected long reads. For pinfish pipeline, we also use minimap2 to mapping the long reads to the reference genome, then “cluster_gff” clusters together the long reads having similar exon/intron. We used “polish_clusters” to creates error corrected long reads based on the cluster definitions generated by “cluster_gff”. Finally, we used “collapse_partials” to collapse identical isoforms to obtain final set of unique, full-length, high-quality isoforms.
Assembly: hg38
Supplementary files format and content: tab-delimited text files include FPKM values for each sample
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| Submission date |
Jun 20, 2022 |
| Last update date |
Aug 02, 2023 |
| Contact name |
Nestor Zhang |
| E-mail(s) |
[email protected]
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| Organization name |
Shanghai Jiao Tong University
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| Street address |
Dongchuan Road
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| City |
Shanghai |
| ZIP/Postal code |
200030 |
| Country |
China |
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| Platform ID |
GPL26167 |
| Series (1) |
| GSE206464 |
Nanopore sequencing combining AlphaFold2 identifies alternative splicing derived putative neoantigens in uveal melanoma |
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| Relations |
| BioSample |
SAMN29208231 |
| SRA |
SRX15798795 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6254978_LR_UM_Case18.txt.gz |
2.9 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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