|
| Status |
Public on Feb 13, 2017 |
| Title |
Luciferase knockdown in DU145 cells using shRNA replicate 3 re-analyzed |
| Sample type |
SRA |
| |
|
| Source name |
brain mets prostate adenocarcinoma
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: prostate adenocarcinoma metastaticized to brain
cancer type: Pca
cell line: DU145
|
| Growth protocol |
DU145 cells were maintained in DMEM media (Mediatech-Cellgro) with 10% FBS, 100 U/ml Penicillin/Streptomycin, and 5microgram/ml puromycin (if selected)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from DU145 cells transduced with shRNA knockdown vector using the Rneasy mini kit (Qiagen) according to manufacturer's instruction
Sequencing libraries were generated using a modified Illumina Truseq sample preparation protocol.Total RNA was DNAase treated with TURBO DNase (Invitrogen) The DNase treated RNA was polyA selected with oligo(dT) beads (Invitrogen). A Superscript III Reverse Transcriptase First-Strand Synthesis (Invitrogen) system was used to generate cDNA from the polyA selected RNA with random hexamer primers (Invitrogen). After first strand synthesis a second strand was generated using E. coli DNA ligase (New England BioLabs) and E. coli DNA polymerase I (New England BioLabs). The double stranded cDNAs were sheared to ~150 nucleotides using a Diagenode BioRuptor and the size was confirmed by DNA gel electrophoresis. DNA end repair of the cDNA was performed using Klenow DNA polymerase (New England BioLabs), T4 DNA polymerase (New England BioLabs), and T4 DNA ligase (New England BioLabs) before the sample was subjected to QIAquick PCR purification (Qiagen). Adapters were ligated to DNA fragments using the T4 DNA ligase (New England BioLabs). The product was run on a 2% agarose gel and size selected between 200 and 300 nucleotides to then be purified by a Gel Extraction kit (Qiagen). Universal and indexing adapters were taken from the TruSeq sample preparation kit (Illumina).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Data processing |
Illumina Casava1.8.2 software used for basecalling
Reads were aligned to the Homo sapien genome (UCSC hg19) using TopHat2 (v2.0.7) running BowTie2 (v2.0.6) and Samtools (v.0.1.18) using a modified protocol from Trapnell et al., Nature Protocols, 2012. The parameters used were -G -p 8
Differential expression of aligned reads was produced using Cufflinks (v2.1.1) using a modified protocol from Trapnell et al., Nature Protocols, 2012. The three replicates for each shRNA DU145 cell line were combined for the differential expression analysis. The parameters used were -b -p 8 -u -L
Genome_build: UCSC hg19
Supplementary_files_format_and_content: tab-delimited text files include for each combination of replicates: RPKM values, locus, status, log2(fold_change), test_stat, p-value, q-value, and significance
|
| |
|
| Submission date |
Aug 30, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Josh Plotnik |
| E-mail(s) |
joshua.plotnik@abbvie.com
|
| Organization name |
AbbVie Inc.
|
| Street address |
1 North Waukegan Rd, AP10/210A
|
| City |
North Chicago |
| State/province |
IL |
| ZIP/Postal code |
60064 |
| Country |
USA |
| |
|
| Platform ID |
GPL11154 |
| Series (2) |
| GSE86239 |
Interaction with ZMYND11 mediates opposing roles of Ras-responsive transcription factors ETS1 and ETS2 |
| GSE86240 |
Interaction with ZMYND11 mediates opposing roles of Ras-responsive transcription factors ETS1 and ETS2 |
|
| Relations |
| Reanalysis of |
GSM1424517 |
| BioSample |
SAMN05716133 |
| SRA |
SRX2067985 |
