11 protocols
Agilent G2565AA and G2565BA Microarray Scanner and Feature ExtractionVersion: 7.0,10.5Agilent publication number: G2566-90017, G4460-90019URL: http://www.chem.agilent.com/Library/usermanuals/Public/G2566-90017.pdf, http://www.chem.agilent.com/Library/usermanuals/Public/G4460-90019_FE_10.5_User.pdfScanner manual for software release 7.0. The Feature Extraction Software User Guide shows you how to set up and run Feature Extraction automatically for a batch of image files and how to extract image files in real time.
Hyridization was carried out according to manufacturer specifications.
Agilent Two-Color Microarray-Based Gene Expression (Quick Amp Labeling)
Version: 5.7
Agilent publication number: G4140-90050
URL: http://www.chem.agilent.com/Library/usermanuals/Public/G4140-90050_Two-Color_GE_5.7.pdf
This document describes Agilent's recommended procedures for the preparation and labeling of complex biological targets and hybridization, washing, scanning, and feature extraction of Agilent 60-mer oligonucleotide microarrays for microarray-based two-color gene expression analysis.
RNA was purified from cells and IP material using Trizol (Invitrogen) according to the manufacters protocol, except with the addition of 1ul of glycogen at the isopropanol precipitation step when purifying IP RNA.
(Parameters: Extracted product = total_RNA, Amplification = none)
CEM cells expressing HA or FLAG-tagged Tat were lysed in Keene lysis buffer (10mM Hepes, 100mM KCl, 5mM MgCl2, 0.5% NP-40 supplemented with 1mM DTT, Complete protease inhibitor and RNaseOUT (100U/ml)) and the nuclei sheared through a 27-guage needle and by freeze-thawing. The lysates were then DNase treated (DNase-turbo, Ambion) on ice for 30 mins, diluted in NT buffer (50mM Tris pH7.5, 150mM NaCl, 1mM MgCl2, 0.05% NP-40, supplemented as before and with 15uM EDTA) and insoluble material removed by centrifugation. Lysates were precleared with protein-G-coated magnetic beads (Dynal) for 1 hour at 4oC and Tat was then immunoprecipitated with anti-HA (3F10; Roche) or anti-FLAG (M2; Sigma) antibody pre-bound to protein-G magnetic beads (Dynal) overnight and the immune complexes washed 4 times with NT2 buffer and twice with NT2 containing 1M urea. Total RNA was then purified from the beads and a sample of total cell lysate with Trizol LS (Invitrogen), DNaseI treated and the integrity verified using an Agilent Bioanalyzer. Using the same protocol, Argo-2 was precipitated with antibodies from Millipore (9E8) or Sigma (11A9); cyclinT1 with an antibody from Santa Cruz (T-18) and CDK9 with C-20 and H-169 (both Santa Cruz).
The T-cell line CEM and stable derivatives expressing GFP from the CMV promoter (CEM-GFP) or HA or FLAG tagged Tat from the HIV LTR (CEM HA-Tat and CEM FLAG-Tat) were cultured in RPMI with 10% FCS and Pen/Strep. The stable cell lines were selected in 500ug/ml Geneticin.
(Parameters: time unit = seconds, min temperature = 37, temperature unit = C, media = RPMI+10% FCS+P/S)
CD4 T-cells were purified from PBMC (Buffy coat) by negative selection (Mitenyl), activated with anti-CD3 and anti-CD28 (1ug/ml) for 60 hours and then expanded in IL2 (5ng/ml) for 48 hours.
(Parameters: time unit = seconds, min temperature = 37, temperature unit = C)
CD4 T-cells were infected with unpurified HIV (NL4-3 strain), NL4-3 purified on a sucrose cushion (both with a dose of 10 infectious units per cell, titred on GHOST cells) or mock-infected by addition of 293-conditioned media. Virus was spinoculated onto the cells at 1000g for 1 hour and infection was allowed to proceed for 1 hour at 37oC. Virus was then washed off the cells and the cells incubated in fresh media.