|
| Status |
Public on Apr 08, 2021 |
| Title |
PC3_ecDNA_negative_En-circles_RNAseq_Rep3 |
| Sample type |
SRA |
| |
|
| Source name |
PC3(CRL1435, ATCC)
|
| Organism |
Homo sapiens |
| Characteristics |
source: The PC3 was initiated from a bone metastasis of a grade IV prostatic adenocarcinoma from a 62-year-old male Caucasian.
ip antibody: NA
|
| Growth protocol |
The circular enhancer DNA (En-circles) or negative control (Ctrl-circles) were purified and pooled at equal molar ratio. 500 ng of En-circles or Ctrl-circles were transfected separately into ecDNA-negative PC3 cells in triplicate using Lipofectamine 3000 (Invitrogen) as per manufacturer’s protocol. Briefly, ecDNA-negative PC3 cells were plated to obtain 70-80% confluence on the following day and were allowed to attach overnight. On the next day, En-circles plasmid DNA was diluted in Opti-MEM reduced serum medium along with P3000 transfection reagent and mixed with Opti-MEM medium containing Lipofectamine 3000. DNA-lipid complexes were allowed to form at room temperature for 15 minutes and mixtures were added to cells. Cells were incubated at standard conditions and harvested 24 hours post-transfection for RNAseq analysis.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Total RNA was isolated in biological replicates using AllPrep DNA/RNA Mini Kit (80204, QIAGEN).
Strand-specific RNA libraries were generated from 300 ng of total RNA following the manuals of KAPA Stranded mRNA Sequencing Kit (KK8502, KAPA Biosystems). Libraries were sequenced on Illumina platform.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
PC3_ecDNA_negative_En-circles_vs_Ctrl-circles.gene_exp.diff
mRNA-seq
|
| Data processing |
The raw sequencing reads were trimmed using Trim Galore version 0.4.3 (options: --stringency 3 -q 20 -e .20 --length 15 --paired) and aligned to the hg19 genome using hisat 2.1.0 (options: --dta-cufflinks). The transcripts were assembled using Cufflinks 2.2.1 and the differential gene expression analysis was performed using Cuffdiff (options: --library-type fr-firststrand).
Illumina RTA-1.18 software used for basecalling
Genome_build: hg19
|
| |
|
| Submission date |
Apr 22, 2020 |
| Last update date |
Apr 08, 2021 |
| Contact name |
Chia-Lin Wei |
| E-mail(s) |
[email protected]
|
| Organization name |
University of Washington
|
| Department |
The Northwest Genomics Center (NWGC)
|
| Lab |
Genome Sciences
|
| Street address |
3720 15th Ave NE
|
| City |
Seattle |
| State/province |
WA |
| ZIP/Postal code |
98195-5065 |
| Country |
USA |
| |
|
| Platform ID |
GPL24676 |
| Series (1) |
| GSE124769 |
Oncogenic extrachromosomal DNA functions as mobile enhancers to globally amplify chromosomal transcription |
|
| Relations |
| BioSample |
SAMN14671419 |
| SRA |
SRX8158072 |
