Genepix 4100a

Images were acquired using GenePix 4100A (Axon Instruments, Molecular Devices Corp., USA) with a 10-μm resolution using the GenePix Pro 6.1 software (Axon Instruments, Molecular Devices Corp., USA). Raw data were processed using Acuity 4.0 (Axon Instruments, Molecular Devices Corp., USA). All the slide data were normalized with a log₂(ratio) average and filtered (signal of each channel > 350 and signal/noise ratio > 2.5). Poor or inconsistent signals were not considered for further analysis. Hybridization signals were depicted as the log2 hybridization signal ratio. Genomic material obtained from three biological independent replicates was used to perform hybridizations two or three times, including a dye-swap, and the genes with signal intensities ± 0.5 were considered for further analysis. FDR<0.05 was used to select significant data. Heat map was generated in MeV 4.9 software using developers instructions were each row represents a specific gene and each column represents each strain. The lightest green boxes are the most underrepresented genes and the brightest red being the most overrepresented genes.

In order to obtain fluorescent images, the dried microarrays were scanned at a 5 µm resolution with an excitation wavelength of 532 nm using the microarray scanner GenePix 4100A (Molecular Devices). Data extraction of the scanned images was performed with the software NimbleScan 2.1 (Roche NimbleGen). The fluorescence intensity values of each microarray were calculated as the average of the replicates of each sequence. For the investigation of sequence dependences background values were subtracted from measured values. The cleavage efficiency was then calculated according to the following equations: $$A=\frac{I_{cleavablesequencesbeforeUDG}-I_{backgroundbeforeUDG}}{I_{controlbeforeUDG}-I_{backgroundbeforeUDG}}$$ $$B=\frac{I_{cleavablesequencesafterUDG}-I_{backgroundafterUDG}}{I_{controlsequencesafterUDG}-I_{backgroundafterUDG}}$$ $$Cleavageefficiency=\left(1-\frac{B}{A}\right)$$
Here I represents the measured fluorescence intensity (in arbitrary units). The consensus sequence logos were generated with WebLogo^{63 (link)} (https://weblogo.berkeley.edu/) based on the list of 5′ to 3′ sequences, containing the variable region only.

A glycan microarray analysis was performed by Ebiogen (Seoul, Korea) according to previous methods [23 (link)]. The Glycan Array kit was purchased from RayBioTech (Norcross, GA, USA). An array containing 300 synthetic glycans printed in quadruplicate on a glass slide was used. Label-based detection was performed according to the manufacturer’s protocols and previous methods [23 (link)]. Biotinylated recombinant lectins and native lectins at 50 μg/mL were added to the array wells and incubated for >3 h with gentle rocking. The glass slide was washed with 1× wash buffer I and II, provided in the kit. Glycan-lectin binding was detected by incubation with Cy3 equivalent dye-conjugated streptavidin for 1 h at room temperature. For cyanine-3 detection, the signals were visualized using a microarray laser scanner (GenePix 4100A; Molecular Devices, Sunnyvale, CA, USA) with excitation at 554 nm and emission at 568 nm. Data extraction was performed using the microarray analysis software, GenePix Pro (ver. 7.2, Sunnyvale, CA, USA). Glycan array data were normalized and analyzed using RayBio Analysis software (RayBioTech, GA-Glycan-300-SW, Norcross, GA, USA).

After synthesis, the nucleobase
and phosphodiester protecting groups
are removed by immersing the slide in the EDA/EtOH solution for 2
h at rt followed by rinsing with deionized water and drying in a microarray
centrifuge. The arrays were then hybridized in a hybridization chamber
(Grace Biolabs) with a mixture consisting of 150 μL of 2×
MES hybridization buffer, 110 μL of nuclease-free water, 13.3
μL of acetylated BSA (10 mg/mL), and 26.7 μL of the 100
nM Cy3-labeled complementary strand at 42 °C for 2 h in a hybridization
oven (Boekel Scientific). After hybridization, the chamber was stripped
off, and the array was washed for 2 min in non-stringent wash buffer
(SSPE; 0.9 M NaCl, 0.06 M phosphate, 6 mM EDTA, 0.01% Tween20), then
1 min in stringent wash buffer (100 mM MES, 0.1 M NaCl, 0.01% Tween20)
followed by a wash in final wash buffer (0.1× SSC) for a few
seconds. The arrays were dried in a microarray centrifuge and then
scanned on a GenePix 4100A microarray scanner (Molecular Devices)
at 5 μm resolution with an excitation wavelength of 532 nm.
Data extraction of the scanned images was performed with NimbleScan
2.1 (NimbleGen), and analysis was performed with Microsoft Excel.

Biotinylated-NGF was prepared using an EZ-Link™ NHS-PEG4-Biotinylation kit (Thermo Fisher scientific, Rockford, IL, USA). About 0.5 mg of NGF (Alomone Labs, Israel) was dissolved in 0.5 mL of phosphate-buffered saline (PBS), mixed with 39 μL of 20 mM biotin solution and incubated at room temperature for 1 h. Excess biotin reagent was removed by a desalting column. The labeled NGF was stored at −80 °C. According to traditional Chinese medicinal theory, around 300 types of CM are related to emotional calmness. These CMs were selected and screened with the biotinylated-NGF probe. The biotinylated-NGF was hybridized with HerboChip dotted with different herbal fractions. Streptavidin-Cy5™ (Invitrogen Life Technologies) was used to detect biotinylated-NGF on the HerboChip arrays. The fluorescence signal of Streptavidin-Cy5 was measured at 535 nm by a fluorophore microarray scanner (GenePix 4100A, Molecular Devices Corp., CA, USA). The fluorescence results were analyzed with GenePix Pro 7 (ver. 7.1.16) software provided by the manufacturer of the microarray scanner (Silicon Valley, CA, USA). Fluorescence intensity higher than 600 was counted as positive.

The 60-mer oligonucleotides custom microarray PINARRAY3 (Cañas et al., 2015 (link)) based on the P. pinaster transcriptome (Canales et al., 2015) was used. Slides were made by Agilent Technologies and hybridization was performed as described by Cañas et al. (2015) (link). Slides were scanned, and signal intensities were recorded using a GenePix 4100A microarray scanner (Molecular Devices, Sunnyvale, CA, USA). Differentially expressed genes (DEGs) were detected using the limma package for R (Smyth, 2005 ). Gene enrichment comparison was performed using the Mapman functional categories through the Mercator web tool (Lohse et al., 2014 ). The microarray data are accessible at NCBI’s Gene Expression Omnibus (Edgar et al., 2002 (link)) through the accession number GSE142093.