Ms200 microarray scanner

The diazotrophic community was analyzed with a microarray-based tool (GeoChip 5.0), which is the latest version of GeoChip. This microarray contains 161,961 probes belonging to 1,447 gene families, including genes involved in crucial biogeochemical processes (e.g., C, N, P, and S cycling) (63 (link)), among which there are 1,331 probes of the nifH gene. For each sample, 1 µg of template DNA was labeled with Cy3 dye, purified with the QIAquick purification kit (Qiagen, Germantown, MD, USA) as previously described (64 (link)), and hybridized with GeoChip 5.0 M microarrays at 67°C with 10% formamide for 24 h. Subsequently, the microarrays were washed, dried, and scanned on an MS 200 microarray scanner (Roche, South San Francisco, CA, USA). Images were quantified into signal intensities with Agilent’s Data Extraction software. Raw signal intensities were uploaded to the Microarray Data Manager of the Institute for Environmental Genomics at the University of Oklahoma (http://ieg.ou.edu/microarray/) for quality control, normalization, and analysis. We normalized the signal intensity of each spot by mean ratio, removed spots with a <2 signal-to-noise ratio (65 (link)), and removed outliers based on standard deviations, as described previously (66 (link)).

Purified DNA was labeled with Cy3, dried, rehydrated, and hybridized with GeoChip 4.0 as described previously54 (link). These arrays were scanned with 100% laser power and a photomultiplier tube using a MS 200 Microarray Scanner (Roche, Basel, Switzerland). The signal intensity of each spot was quantified using ImaGene software (Arrayit, Sunnyvale, CA, USA), and poor-quality spots with a signal/noise ratio (SNR) < 2.0 were discarded prior to statistical analyses54 (link).
Raw GeoChip data were uploaded to the GeoChip data analysis manager (http://ieg.ou.edu/microarray/) and pre-processed using the data analysis pipeline as previously described54 (link). The following major steps were performed: (i) removal of poor quality spots (SNR < 2.0); (ii) normalization of the signal intensity of each spot based on the mean signal intensity across all genes on the arrays; and (iii) transformation of the data using the natural logarithmic form. Pre-processed GeoChip data were used for further statistical analysis.

A double-stranded cDNA synthesis kit (InvitroGen) was used to produce cDNA from 2 μg of total RNA. cDNA (1 μg) was labeled using the one color DNA labeling kit (Nimblegen – Roche) and labeled cDNA (2 μg) was hybridized onto E. coli K-12 gene expression arrays (Nimblegen – Roche) for 17 h at 42 °C according to the manufacturers’ instructions. Arrays were washed and then scanned with a MS200 Microarray Scanner (Nimblegen – Roche). The images were analyzed with DEVA 1.2.1 software. Only raw data were used for further analyses. All array procedures were performed by the GeT-Biopuces platform (http://get.genotoul.fr).

The hybridized arrays were scanned on an MS200 microarray scanner (Roche, Basel, Switzerland) at a resolution of 2 µm. The raw methylation data were extracted with the default ChIP protocol from software NimbleScan for 385 K arrays and with DEVA for 2.1 M arrays. Methylation ratios between the IP DNA samples and the control input samples were normalized across samples using the quantile method after performing a variance stabilization using log2 scaling for each promoter feature on the array. All data processing including mapping of microarray probes to promoters, gene annotation, data post-processing, principal component analysis, identification of differentially methylated regions, and graphics were performed with in-house developed functions in Matlab^{51 (link),52 (link)}.

Mycelial samples from the one litre bioreactors were collected by vacuum filtration on Whatman GF/A filter paper and then rinsed in 0.9% NaCl and frozen immediately in liquid nitrogen. RNA was isolated from 50 mg of the mycelia with RNeasy Plant RNA isolation kit (Qiagen GmbH. Hilden, Germany). RNA quality was assessed for purity and quantity using an Agilent RNA 6000 analyser (Agilent Technologies Inc. Santa Clara, CA, United States of America) The purified RNA was then converted into cDNA using an Invitrogen Superscript Double-Stranded cDNA Synthesis Kit (Thermo) prior to labelling with Cy3 One-Colour fluorescent dye (Roche NimbleGen Inc. Basel, Switzerland) according to the manufacturer’s instructions. The labelled cDNA was hybridised on an automated HS12 system (Roche) to custom-made four plex microarray slides (Roche) designed from the T. reesei genome version 2.0 [23 (link),24 (link)]. Analysis was performed in an MS200 microarray scanner (Roche) to identify signal probe intensity of the six 50-74mer probe replicates designed from the known and predicted genes in T. reesei.

Macaque serum samples were diluted 1:100 in binding buffer (0.01M Tris-Cl, pH 7.4, 1% alkali-soluble casein, 0.05% Tween-20). Diluted sample aliquots and binding buffer-only negative controls were bound to arrays overnight for 16–20 h at 4°C. After binding, the arrays were washed 3x in wash buffer (1x TBS, 0.05% Tween-20), 10 min per wash. Primary sample binding was detected via 8F1-biotin mouse anti-primate IgG (NIH Nonhuman Primate Reagent Resource) secondary antibody. The secondary antibody was diluted 1:10,000 (final concentration 0.1 ng/µl) in secondary binding buffer (1x TBS, 1% alkali-soluble casein, 0.05% Tween-20) and incubated with arrays for 3 h at room temperature, then washed 3x in wash buffer (10 min per wash) and 30 sec in reagent-grade water. The secondary antibody was labeled with Cy5-Streptavidin (GE Healthcare; 5 ng/µl in 0.5x TBS, 1% alkali-soluble casein, 0.05% Tween-20) for 1 h at room temperature, then the array was washed 2x for 1 min in 1x TBS, and washed once for 30 sec in reagent-grade water. Fluorescent signal of the secondary antibody was detected by scanning at 635 nm at 2 µm resolution and 25% gain, using an MS200 microarray scanner (Roche NimbleGen).