 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Jul 31, 2022 |
| Title |
H358-CDDP |
| Sample type |
SRA |
| |
|
| Source name |
Non-small cell lung cancer cell line
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: H358
treatment: Cisplatin
extract: Total cells
|
| Treatment protocol |
Treatment with cisplatin (Selleckchem, Euromedex, Souffelweyersheim, France) was done at 100 µM. Treatment with vehicle (DMSO) was done in parallel.
|
| Growth protocol |
Cells were cultured in RPMI-1640-Glutamax medium (GibcoBRL, Life Technologies, Cergy Pontoise, France), supplemented with 10% (v/v) heat-inactivated fetal calf serum (GibcoBRL), in 5% CO2 at 37°C.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
RNA was extracted from total cells.
A starting amount of 1 µg was used for long read library preparation. The first step of cDNA synthesis was done using the SMARTer PCR cDNA Synthesis Kit (Takara). Then, cDNA was amplified by 14 PCR cycles and divided in two fractions to be purified with different AMPure PB beads (Pacific Biosciences) ratio. In order to enrich for larger transcripts, a ratio of 0.4x was applied to a fraction (corresponding to 5 out of 8 of the initial volume), while the other fraction was purified with 1x ratio. For each sample, both fractions were quantified with Qubit dsDNA high-sensitivity kit (ThermoFisher Scientific), qualified by capillary electrophoresis (Bioanalyzer High Sensitivity DNA kit - Agilent), pooled equimolarly, and subjected to the SMRTbell Template Prep Kit 1.0 (Pacific Biosciences). The first DNA damage and end repair steps were applied according to the manufacturers’ recommendations. The following ligation was extended to 16 hours to improve efficiency. The final exonuclease treatment was applied to digest unwanted molecules. The SMRTbells were quantified with Qubit dsDNA high-sensitivity kit (ThermoFisher Scientific) and qualified by capillary electrophoresis as above. Complexes were prepared following the manufacturer’s recommendations using the Sequel Binding Kit 3.0 and the sequencing Primer v3 (Pacific Biosciences). Each sample was sequenced on 4 SMRTcells 1M, on the Sequel I system (Pacific Biosciences) using a loading molarity of 5 pM. For each SMRTcell, 10 hours of movie, 2 hours of immobilization, and 4 hours of pre-extension were set up.
|
| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Sequel |
| |
|
| Description |
replicate 1
|
| Data processing |
Library strategy: ISO-seq
Subreads coming from PacBio SEQUEL SMRTcells were processed via the SMRTLink’s IsoSeq v3.0 pipeline with default parameters (https://github.com/PacificBiosciences/IsoSeq/blob/master/isoseq-clustering.md). This pipeline is composed of the following 6 steps: Circular Consensus Sequence (CCS) calling, primer removal/demultiplexing, refining, merging, clustering and polishing. Resulting CCS reads and HQ transcripts were mapped with minimap2 (v2.17-r941) against hg19 genome with the following parameters : -ax splice -uf --secondary=no -C5 -O6,24 -B4.
Then, CCS reads were classified into 'last exon (LE)' and 'intronic polyadenylation (IPA)' isoforms, depending on whether the 3'-end of the read was located in the last exon of a gene or within a gene but upstream of the last exon ('ELE' region).
Genome_build: hg19
Supplementary_files_format_and_content: .xlsx
|
| |
|
| Submission date |
Jul 27, 2021 |
| Last update date |
Jul 31, 2022 |
| Contact name |
Céline M. Labbé |
| E-mail(s) |
celine.labbe@curie.fr
|
| Organization name |
Institut Curie
|
| Street address |
Rue Henri Becquerel
|
| City |
Orsay |
| ZIP/Postal code |
914001 |
| Country |
France |
| |
|
| Platform ID |
GPL26180 |
| Series (2) |
| GSE180918 |
Cisplatin-induced decrease of mRNA length reveals functional and translated intronic-polyadenylation isoforms in the annotated 5’-untranslated region of genes [ISO-seq] |
| GSE180930 |
Cisplatin-induced decrease of mRNA length reveals functional and translated intronic-polyadenylation isoforms in the annotated 5’-untranslated region of genes |
|
| Relations |
| BioSample |
SAMN20434363 |
| SRA |
SRX11569346 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5478418_CCS_H358_CDDP_IPA.xlsx |
26.0 Mb |
(ftp)(http) |
XLSX |
| GSM5478418_CCS_H358_CDDP_LE.xlsx |
121.4 Mb |
(ftp)(http) |
XLSX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
|
|
|
|
 |
