Genepix 4000b scanner

Because of the early initiation time of this study, we could only collect 193 miRNAs, and the identities were updated using the latest version of the miRBase database (version 21). The mature miRNA sequences and six control probes (four positive and two negative) were used to produce the customized rice miRNA microarray (Combimatrix Custom Array 4 × 2 K, CA, USA). Each miRNA probe was supplied in triplicate on the microarray, and each control probe contained five copies. For each rice cultivar, 2 μg of the purified small RNAs was employed to prepare the fluorochrome-labeled miRNAs (Cy5 Labeling Kit; Mirus Bio LLC, Madison, WI, USA) according to the manufacturer's instructions. Subsequently, the customized miRNA microarray was hybridized with the Cy5-labeled miRNAs in a 42ºC oven with slow microarray rotation for 4 h. After hybridization, the microarray was washed with a SSPE buffer series with 0.05% Tween-20 according to the manufacturer’s protocol (Combimatrix) and then subjected to image scanning and digitization using a GenePix 4000B scanner and GenePix 4.0 software (Molecular Devices) for further data analysis.

DNA microarray analyses were performed for the four groups, i.e., A1, A2, A5, and A7. After cDNA synthesis, Cy3-labeled cRNA was synthesized and purified using the Low Input Quick Amp Labeling Kit (Agilent) according to the manufacturer’s instructions. Notably, reverse transcription was conducted using a T7 promoter-oligo(dT) primer. Absorbance was measured at 260, 280, 320, and 550 nm, and it was verified that the labeled cRNA had incorporated >6 pmol/mg of Cy3-CTP. Labeled cRNA was fragmented using the Gene Expression Hybridization Kit (Agilent) and applied to Whole Mouse Genome Array Ver2.0 slides (Agilent). After hybridization at 65 °C for 17 h, the slides were washed with Gene Expression Wash Buffers 1 and 2 (Agilent) according to the manufacturer’s instructions. The slides were scanned using a GenePix 4000B scanner (Molecular Devices Japan K.K., Tokyo, Japan). Scanned images were digitalized and normalized using GebePix Pro software (Molecular Devices Japan K.K., Tokyo, Japan).

The customized 4 × 44 K oligoarray (Agilent Technologies, Santa Clara, U.S.A.) for sugarcane (CaneRegNet) contains 21,901 unique probes in duplicates, representing 14,522 SAS released by the Sugarcane EST Project (SUCEST)74 (link) and designed as described by Lembke et al.75 (link). All the steps (from sample preparation to hybridization) followed the Two-Color Microarray-Based Gene Expression Analysis protocol. Spike-in RNA that hybridize to control probes in the array was added to monitor the workflow (Two-Color RNA Spike-In Kit, Agilent Technologies, Santa Clara, U.S.A.). Amplification and labelling (cyanine 5-CTP and cyanine 3-CTP dyes) of 2 μg of total RNA to generate cRNA were performed using Quick Amp Labelling kit, two-color (Agilent Technologies, Santa Clara, U.S.A.). Then, cRNA was purified by RNeasy Mini kit (Qiagen, Hilden, Germany) and quantified in NanoDrop ND-1000 UV-VIS spectrophotometer (Thermo Scientific, Life Technologies, Waltham, U.S.A.). Fluorescently-labelled cRNA was hybridized to arrays using the Gene Expression Hybridization kit and the Gene Expression Wash Buffer kit (Agilent Technologies, Santa Clara, U.S.A.). The arrays were scanned by GenePix 4000B scanner (Molecular Devices, Sunnyvale, U.S.A.) using Agilent (C or B) Scanner Settings. Microarray data files are deposited at the Gene Expression Omnibus (GEO) public database, series record GSE85489.

Binding of recombinant CH103 Ab mutants to 9,400 human proteins was determined using a ProtoArray (Invitrogen, Waltham, MA) in duplicate, according to the manufacturer instructions and as described previously (13 (link), 23 (link), 50 (link), 51 (link)). In brief, protein microarrays were blocked with 2 µg/ml of recombinant Abs or 151K, an isotype-matched (IgG1, κ) human myeloma control Ab (Southern Biotech) for 1.5 hours at 4°C. Ab binding to array proteins was detected using 1 µg/ml of Alexa Fluor 647-conjugated anti-human IgG (Invitrogen), using mild agitation at 4°C for 1.5 hours. Microarrays were scanned using a GenePix 4000B scanner (Molecular Devices, San Jose, CA). Fluorescence intensities of Ab binding to each protein were quantified by aligning image data with the GenePix Pro 5.0 program (Molecular Devices) using lot-specific protein spot definitions provided by the manufacturer, and by comparing Ab-binding patterns to the 151K control.

Total RNAs from wheat leaves were extracted using the RNeasy Plant Mini Kit (Qiagen, Courtaboeuf, France). The quality of RNAs was measured with Nanodrop by analyzing their absorbance ratios, A260/280 and A260/230, which were found to range between 2 and 2.2. Besides, RNA quality was also examined with Bioanalyzer 2100 (Agilent, France), and a minimal RNA integrity number (RIN) of 8 was required for all the samples. A non-targeted gene expression analysis was performed using a Microarrays GE 4x44 chip purchased from Agilent (Santa Clara, CA, United States). The hybridization for all the conditions was carried out in triplicate, with each replicate generated using the total RNA extracted from three bulked leaves harvested from different pots (nine pots were used in total). All steps of RNA amplification, staining, hybridization, and washing were performed according to the manufacturer's indications. Slides were scanned at 5 μm/pixel resolution using a GenePix 4000B scanner (Molecular Devices Corporation, Sunnyvale, CA, United States), and the images were used for grid alignment and expression data digitization using the software GenePix Pro 6.0 (Molecular Devices Corporation, Sunnyvale, CA, United States). Transcriptomics data have been submitted to the NCBI GEO Archive for Functional Genomics Data with the accession number GSE178704.

Oligoarray sample preparation and analysis was performed as described in Lembke et al. [20 (link)], using two biological replicates for control and treated samples from: (i) 4 or 6 days of water privation, and 2 days of re-watering (greenhouse-grown plants); and (ii) 7 months after planting (field-grown plants). Only RNA samples with RIN ≥ 6.5 were selected, and hybridizations were performed using the customized sugarcane Agilent oligoarray platform [20 (link)]. The arrays contained probes to detect the sense and antisense expression of 14,522 different Sugarcane Assembled Sequences (SAS), which are reference sequences for sugarcane transcripts [42 (link)]. Hybridized oligoarray slides were scanned in GenePix 4000B scanner (Molecular Devices, San Jose, CA, USA), and image data were extracted with the aid of the Feature Extraction 9.5.3 (Agilent Technologies, Santa Clara, CA, USA) using the two-color oligoarray referential in the Agilent platform intensity [105 (link)] from Cy3 and Cy5 was corrected and normalized using the Lowess function [106 (link)] implemented in the R software. Microarray data files are deposited at the Gene Expression Omnibus (GEO) public database, series record GSE125069.