|
| Status |
Public on Apr 19, 2022 |
| Title |
PANC1 CID total rep 4 |
| Sample type |
SRA |
| |
|
| Source name |
pancreatic ductal adenocarcinoma
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: Human pancreatic ductal adenocarcinoma cell line
cell line: PANC1
treatment: CID661578 (40 uM), 6hrs
sample identifier: P22951_1041
replicate: 4
molecule subtype: total cytosolic RNA
|
| Treatment protocol |
Four million PANC1 cells were seeded in 15 cm plates 24 hours prior to treatment prior to treatment with CID661578 (40 uM), cercosporamide (5 uM), or DMSO. Treatments were administered in complete growth media, and were applied to cells for 6 hours.
|
| Growth protocol |
PANC1 cells were cultured in RPMI 1640 media with GlutaMAX, supplemented to a final concentration of 10% Fetal Bovine Serum (FBS, Gibco), and penicillin (100 units/mL)/streptomycin (100 ug/mL). Cells were kept in a humidified incubator at 37°C in 5% CO2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Polysome profiling was performed on four replicates of PANC1 cells treated with MNK2 inhibitors or DMSO as previously described (Ristau et al. Methods Mol Biol, 2022). Briefly, total cytosolic RNA was isolated from aliquots of cell lysates using TRIzol reagent. The remaining lysates were layered onto optimized sucrose density gradients (5:34:55% [w/v]) allowing polysomes to be fractionated. Fractions corresponding to mRNAs associated with more than three ribosomes were collected in TRIzol reagent and pooled, allowing isolation of efficiently translated polysome-associated mRNA.
Smart-seq2 RNA sequencing libraries were prepared as previous described (Picelli et al. Nature Protocols, 2014) using 10 ng of RNA input per sample. Pre-amplification PCR was performed with 12 cycles, and 100 pg of cDNA was used for tagmentation. After tagmentation, adapter-ligated fragments were amplified with 14 PCR cycles.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Data processing |
The Bcl to FastQ conversion was performed using bcl2fastq_v2.20.0.422 from the CASAVA software suite. The quality scale used is Sanger / phred33 / Illumina 1.8+.
RNA sequencing read quality was assessed using FastQC v.0.11.8 and MultiQC.
Sequencing adapters and reads mapping to ribosomal RNA were removed using BBDuk from the BBTools suite (http://jgi.doe.gov/data-and-tools/bb-tools/) prior to alignment to hg38 using HISAT2 with default settings.
Reads were summarized using the featureCounts function of the Rsubread R package with default settings and RefSeq gene definitions.
Assembly: GRCh38
Supplementary files format and content: Matrix of raw gene expression counts based on RefSeq gene definitions.
|
| |
|
| Submission date |
Apr 08, 2022 |
| Last update date |
Apr 19, 2022 |
| Contact name |
Ola Larsson |
| E-mail(s) |
ola.larsson@ki.se
|
| Phone |
+46 (0)8 517 73280
|
| Organization name |
Karolinska Institutet
|
| Department |
Department of oncology-pathology
|
| Street address |
CCK, R8:01
|
| City |
Stockholm |
| ZIP/Postal code |
171 76 |
| Country |
Sweden |
| |
|
| Platform ID |
GPL24676 |
| Series (1) |
| GSE200477 |
Polysome profiling quantified by RNA sequencing in PANC1 cells treated with MNK2 inhibitors or DMSO |
|
| Relations |
| BioSample |
SAMN27478544 |
| SRA |
SRX14786722 |
