|
| Status |
Public on Nov 30, 2015 |
| Title |
neg.co_d1_3 |
| Sample type |
SRA |
| |
|
| Source name |
peripheral blood monocytes
|
| Organism |
Homo sapiens |
| Characteristics |
transfection: Control siRNA
|
| Treatment protocol |
Purified monocytes were transfected with Stabilin-1 siRNA and negative control siRNA. The cells were then cultured for 24 hours under non-polarizing culture medium. Stabilin-1 expression was determined by FACS from aliquots of cells to verify the silencing efficacy.
|
| Growth protocol |
Blood samples from healthy adults were diluted with HBSS and laid over the Ficoll gradient and centrifuged at 2000 rpm for 20 min and 20oC. The leukocyte containing interface was collected and washed with HBSS three times by centrifuging the cells at 1200 rpm for 10 min at 10oC. Monocytes were enriched using MACS negative selection kit (Monocyte isolation Kit II with CD16) following the manufacturer’s protocol.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted from the transfected monocytes using the Nucleo-Spin RNAII Total RNA Isolation Kit (Macherey-Nagel), and the quality of the isolated RNA was checked with the Agilent 2100 Bioanalyzer (Agilent Technologies).
RNA libraries were prepared for sequencing using standard Illumina protocols.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Data processing |
Basecalls performed using the instrument manufacturer’s (Illumina HiSeq2500) Bcl2fastq version 1.8.4 base calling software
the reads were aligned against the human reference genome (hg19 assembly, downloaded from UCSC) using TopHat version 2.0.10
Reads were associated with known genes based on RefSeq annotations derived from UCSC database, the number of associated reads was counted using HTSeq tool (v 0.5.4p3)
Counts were normalized using the TMM normalization method of the edgeR R/Bioconductor package.
For statistical analysis the data were further transformed using the voom approach in the limma package.
R package Limma was used for performing the statistical testing between the groups
Filtering of differentially expressed genes was done using the following thresholds: FC>2.0, p<0,05
Supplementary_files_format_and_content: tab-delimited text files include RPKM values with annotations for each sample
|
| |
|
| Submission date |
Dec 03, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Kati Elima |
| E-mail(s) |
kati.elima@utu.fi
|
| Organization name |
University of Turku
|
| Department |
MediCity
|
| Street address |
Tykistökatu 6
|
| City |
Turku |
| ZIP/Postal code |
20520 |
| Country |
Finland |
| |
|
| Platform ID |
GPL16791 |
| Series (1) |
| GSE63807 |
Transcriptome of Stabilin-1 siRNA transfected human monocytes |
|
| Relations |
| BioSample |
SAMN03251966 |
| SRA |
SRX794227 |
