|
| Status |
Public on Mar 22, 2018 |
| Title |
D186_HIV_8h |
| Sample type |
RNA |
| |
|
| Source name |
monocyte-derived dendritic cells infected with HIV-GFP in the presence of Vpx, time (hours): 8, drug: none, donor: D186
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: monocyte-derived dendritic cells
|
| Treatment protocol |
Monocyte-derived dendritic cells that had been treated with virus-like particles packaging Vpx were infected with HIV-GFP in the presence or absence of Raltegravir (25 uM) in a time course according to the following protocol: On day 4 after differentiation from monocytes, dendritic cells were counted and resuspended at 800,000 cell per ml in fresh DC medium with GM-CSF, IL-4, and polybrene (1 ug/ml) and then plated into round bottom 96 well plates with 75 ul per well. Cells were mock-treated or treated with Raltegravir and then infections were performed by diluting virus in fresh medium (without cytokines or polybrene) to a final volume normalized to controls (160 ul per well). The time course was performed in reverse order such that all samples were harvested at the same time on day 6 after DC differentiation. Mock-infected samples were treated with Vpx and DC media in parallel with samples that received HIV-GFP for 40 hours.
|
| Growth protocol |
To generate monocyte-derived dendritic cells, CD14+ monocytes from PBMC buffy coats were isolated with anti-human CD14 magnetic beads (Miltenyi) and cultured in RPMI containing 10% heat-inactivated fetal bovine serum (FBS), 50 U/ml penicillin, 50 ug/ml streptomycin (P/S), 10 mM HEPES, beta mercaptoethanol, and 2 mM glutamine, in the presence of recombinant human GM-CSF at 10 ng/ml and IL-4 at 50 ng/ml (Peprotech). To overcome restriction to HIV-1 reverse transcription, virus-like particles packaging Vpx were added to isolated cells on day 0. Fresh media and cytokines were added to cells (40% by volume) on day 1. On day 4, cells were collected, resuspended in fresh media with cytokines used for infection.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated with TRIzol (ThermoFisher) according to the manufacturer's instructions using two sequential chloroform extractions and adding Glycoblue as carrier prior to precipitation.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 100-200ng total RNA using Agilent’s Low Input Quick Amp One-Color Labeling Kit according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
600ug of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 25ul containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturer’s instructions. On completion of the fragmentation reaction, 25ul of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent SurePrint G3 8x60K Microarrays using the Agilent hybridization chambers. The hybridization was allowed to run for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried by compressed air.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505C) using one color scan setting for 8x60k array slides (Scan Area 61mmx21.6mm, Scan resolution 3um, Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
Gene expression of human monocyte-derived DCs 8 hours after infection with HIV-GFP in the presence of virus-like particles packaging Vpx
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.7.3.1 (Agilent) using default parameters (protocol GE1_107_Sep09 and Grid:039494_D_F_20120628. Probe sequences were mapped against the Ensembl transcript database (ensembl.org, GRCh37.74). Only sequences that matched with at most 5 mismatches and were best matches were retained. Sequences that mapped to more than one gene were removed for a total of 37,623 unique probe sequences. Probe-specific logarithmically transformed expression was quantile-normalized. Duplicate probe sequences were averaged.
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| |
|
| Submission date |
Jun 22, 2017 |
| Last update date |
Mar 22, 2018 |
| Contact name |
Jarrod S Johnson |
| Organization name |
Center for Infectious Disease Research
|
| Street address |
307 Westlake Ave, Suite 500
|
| City |
Seattle |
| State/province |
Washington |
| ZIP/Postal code |
98109 |
| Country |
USA |
| |
|
| Platform ID |
GPL17077 |
| Series (2) |
| GSE100374 |
HIV-1 infection of human monocyte-derived dendritic cells with and without the integrase inhibitor, Raltegravir (gene expression). |
| GSE100377 |
HIV-1 infection of human monocyte-derived dendritic cells with and without the integrase inhibitor, Raltegravir |
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