|
| Status |
Public on Jul 31, 2016 |
| Title |
siProxSC_III.bam |
| Sample type |
SRA |
| |
|
| Source name |
CGTH-W-1_PROX1 siRNA (Scruz)
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: CGTH-W-1
cell type: follicular thyroid carcinoma cells
treated with: PROX1 siRNA (Scruz)
|
| Treatment protocol |
Cells were transfected either with non-targeting MISSION® siRNA Universal Negative Control (Sigma Aldrich, cat. no. SIC001) [siNEG-samples] or two PROX1 specific siRNA: 1) Sigma-Aldrich - cat. no. EHU053851 [siProxSig-samples] and 2) Santa Cruz - cat. no. sc-106451 [siProxSC-samples] in a presence of Lipofectamine 2000 (LifeTechnologies, cat. no.11668-019). Treatment of cells with Lipofectamine 2000 alone was also performed [lipo-samples]. 48 hours later cells were harvested.
|
| Growth protocol |
CGTH-W-1 cell line (German Collection of Microorganisms and Cell Cultures, DSMZ no. ACC 360) was cultured in RPMI-1640 supplemented with 10% FBS (Gibco).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated with GeneMATRIX Universal RNA Purification Kit, (cat. no. E3598, EURx), and RNA integrity was assessed using Agilent RNA 6000 Nano Kit on Agilent 2100 Bioanalyzer. All transfections were performed in three independent replicates, numbered I, II and III. Efficacy of PROX1 knockdown was measured with RT-qPCR using specific primers (forward 5'-CCAGCTCCAATATGCTGAAGACCTA-3'; reverse 5'-CATCGTTGATGGCTTGACGTG-3') and Sybr Green chemistry.
RNA was subjected to library preparation using Ion AmpliSeq Transcriptome Human Gene Expression Panel, according to manufacturer's protocol. Briefly, RNA was reverse transcribed and cDNA subjected to multiplex PCR reaction to amplify parts of targeted transcripts. Amplicons were then partially digested at primer sequences followed by adapters ligation to amplicons and purification on AMPure® XP beads. Resulting library was quantified on Bioanalyzer 2100 and diluted to ~100 pM prior to template preparation. Up to eight barcoded libraries were subjected to automated template preparation with Ion PI IC 200 Kit on the Ion Chef Instrument, which performs emulsion PCR on Ion Sphere particles (ISPs), followed by ISPs recovery and template loading on a PI chip. Barcoded RNA-Seq libraries samples were sequenced on a PI chip using the sequencing reagents provided as part of the Ion PI IC 200 Kit, according to the manufacturer’s instructions.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Ion Torrent Proton |
| |
|
| Data processing |
Raw reads were processed by Torrent Suite analysis pipeline and mapped to human genome assembly hg19 AmpliSeqTranscriptome version by TMAP.
Reads corresponding to each gene were counted with htseq-count (PMID:25260700).
Normalization and differential expression were conducted by DESeq2 (PMID:25516281), using default parameters and options.
Genome_build: hg19
Supplementary_files_format_and_content: Comma-separated values file with DESeq2 output for each gene comparing expression in two groups
|
| |
|
| Submission date |
Apr 11, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Katarzyna Paczkowska |
| E-mail(s) |
paczkowska.km@gmail.com
|
| Organization name |
Cancer Center - Institute
|
| Department |
Department of Genetics
|
| Street address |
Roentgena 5
|
| City |
Warsaw |
| ZIP/Postal code |
02-781 |
| Country |
Poland |
| |
|
| Platform ID |
GPL17303 |
| Series (1) |
| GSE80135 |
Prox1 (Prosper-related homeobox 1) transcription factor depletion effect on CGTH-W-1 cells. |
|
| Relations |
| BioSample |
SAMN04632359 |
| SRA |
SRX1689725 |
