|
| Status |
Public on Jul 14, 2023 |
| Title |
NCI-H1975 + sequins mixA mixed with HCC827 + sequins mixB in ratio 0:100 |
| Sample type |
SRA |
| |
|
| Source name |
Lung adenocarcinoma
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: Lung adenocarcinoma
cell line: NCI-H1975, HCC827
|
| 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).
The NCI-H1975+sequins mixA replicate 1 RNA was mixed with the HCC827+sequins mixB replicate 1 RNA in the 5 following mass ratios: 100:0, 75:25, 50:50, 25:75 and 0:100. 1 ng of each mix was then used for library preparation. The cRNA-PCR Barcoding (SQK-PCS109 with SQK-PBK004) kit was used with the supplied ONT protocol. Amplication for purified mRNA spiked with sequins was done with 16 PCR cycles with a 2 minutes and 30 seconds extension step and a 0.8X final bead clean up. Size selection was performed for the standard ~2kb target. 65 ng of the final library were loaded onto 1 FLO-PRO002 PromethION flow cell (R9.4.1).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
PromethION |
| |
|
| Data processing |
Reads were basecalled with Guppy 6.2.1 in `sup’ accuracy (dna_r9.4.1_450bps_sup_prom.cfg), disabling trimming and enabling read splitting.
Passed reads were aligned to the Gencode V33 reference transcriptome and RNA sequin annotation 2.4 with minimap2 version 2.24.
Transcript-level counts were obtained using salmon version 1.9.0.
Assembly: GrCh38 and RNA sequin annotation 2.4
Supplementary files format and content: Tab-separated files with transcript IDs, lengths and read counts
|
| |
|
| Submission date |
Mar 09, 2023 |
| Last update date |
Jul 15, 2023 |
| Contact name |
Matthew Ritchie |
| E-mail(s) |
mritchie@wehi.edu.au
|
| 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) |
| GSE227000 |
Long-read transcriptome profiling of human lung cancer cell lines |
|
| Relations |
| BioSample |
SAMN33698269 |
| SRA |
SRX19610930 |
