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Sample GSM2679703 Query DataSets for GSM2679703
Status Public on Mar 22, 2018
Title D187_VLP_mock
Sample type RNA
 
Source name monocyte-derived dendritic cells with mock infection in the presence of Vpx, time (hours): 0, drug: none, donor: D187
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 after mock infection 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.
 
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

Data table header descriptions
ID_REF
VALUE Normalized log2-transformed signal intensity

Data table
ID_REF VALUE
A_23_P117082 11.3331175131078
A_33_P3246448 5.54592976908547
A_33_P3318220 5.28771237954945
A_33_P3236322 5.50283180406674
A_33_P3319925 5.88264304936184
A_21_P0000744 6.72643105611424
A_24_P215804 5.04507705193494
A_23_P110167 10.6414857452827
A_33_P3211513 7.32699117490082
A_23_P103349 5.15748158370491
A_32_P61480 4.77066389291676
A_33_P3788124 4.6838718686226
A_33_P3414202 8.39090785252409
A_33_P3316686 8.54006748093771
A_33_P3300975 5.53186877517755
A_33_P3263061 9.81343049355114
A_33_P3261373 5.13057056280543
A_24_P278460 8.14003089205449
A_21_P0014651 6.31174831500496
A_24_P286898 5.88264304936184

Total number of rows: 37578

Table truncated, full table size 1110 Kbytes.




Supplementary file Size Download File type/resource
GSM2679703_20150630_D187_VLP_mock.txt.gz 12.3 Mb (ftp)(http) TXT
Processed data included within Sample table

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