|
| Status |
Public on Jul 20, 2022 |
| Title |
R2_HCC827_ONT |
| Sample type |
SRA |
| |
|
| Source name |
Human lung cancer cell line HCC827
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: Human lung cancer cell line
cell line: HCC827
replicate: 2
|
| Treatment protocol |
Cells were dissociated into single cell suspensions in FACS buffer, centrifuged and frozen at -80 °C.
|
| Growth protocol |
The cell lines were retrieved from the ATCC (https://www.atcc.org/) and cultured in Roswell Park Memorial Institute (RPMI) 1640 medium with 10% fetal calf serum and 1% penicillin-streptomycin. The cells were grown independently at 37°C with 5% carbon dioxide until near 100% confluency.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The mRNA was extracted using a Qiagen RNA miniprep kit and purified using the NEBNext® Poly(A) mRNA Magnetic Isolation Module (E7490).
3.75ng purified mRNA spiked with sequins were used for the NEBNext Ultra II Directional RNA Library Prep Kit (Illumina). Fragmentation was done for 15 minutes at 94°C, and final amplification with 11 PCR cycles. The libraries were sequenced in 75PE on NextSeq 500. The cRNA-PCR Barcoding (SQK-PCS109 with SQK-PBK004) kit was used with the supplied protocol (ONT). Amplication for 1.5ng purified mRNA spiked with sequins was done with 14 PCR cycles with a 2 minutes and 30 seconds extension step. Pooled barcoded libraries were sequenced on 5 PromethION R9 flow cells. Iso-Seq libraries were prepared and sequenced by Novogene on Sequel II (PacBio). Libraries were prepared according to the manufacturer's specifications from total RNA spiked in with sequins. Size selection was performed for the standard ~2kb target. The total number of PCR cycles is 12.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
PromethION |
| |
|
| Data processing |
MinKNOW PromethION Release 19.12.9 (ONT)
Base calling and quality scoring (Illumina): Real-Time Analysis on board software v2.4.6. Base calling (ONT): Guppy 4.2.2, with the PromethION model, in high-accuracy mode.
Circular consensus sequence generation (PacBio): ccs 4.0.0 (commit SL-release-8.0.0).
FASTQ file generation and de-multiplexing (Illumina): bcl2fastw conversion software v2.15.0.4. De-multiplexing (PacBio): lima 1.10.0 (commit SL-release-8.0.0).
Refining and clustering (PacBio): isoseq3 3.2.2 (commit SL-release-8.0.0)
Sequenced reads were mapped to the GRCh38 reference genome and RNA Sequin decoy chromosome combined sequences using the align function in the Rsubread package (version 1.34.7, Liao et al. Nucleic Acids Research, 2013) with default parameters (Illumina), minimap2 version 2.17 with arguments -ax splice MD -L (ONT) and pbmm2 1.1.0 (commit SL-release-8.0.0) (PacBio).
Gene-level counts were obtained using the featureCounts function (Liao et al. Bioinformatics, 2014) from the Rsubread package (version 1.34.7) using the GENCODE v33 annotation and RNA sequin annotation 2.4 combined GTF file (Illumina) and the GENCODE v29 annotation and RNA sequin annotation 2.4 combined GTF file (ONT, PacBio) .
Genome_build: GrCh38 and RNA sequin decoy chromosome 2.4 combined sequences
Supplementary_files_format_and_content: Tab-delimited text files with columns representing Ensembl IDs and the number of reads mapping to that gene.
|
| |
|
| Submission date |
Apr 20, 2021 |
| Last update date |
Jul 20, 2022 |
| Contact name |
Matthew Ritchie |
| E-mail(s) |
[email protected]
|
| Organization name |
The Walter and Eliza Hall Institute of Medical Research
|
| Street address |
1G Royal Parade
|
| City |
Parkville |
| State/province |
Victoria |
| ZIP/Postal code |
3052 |
| Country |
Australia |
| |
|
| Platform ID |
GPL26167 |
| Series (1) |
| GSE172421 |
Long and short-read transcriptome profiling of human lung cancer cell lines |
|
| Relations |
| BioSample |
SAMN18810602 |
| SRA |
SRX10644320 |
