Microarray scanner

Microarray hybridization, nanoLC-MS/MS analysis and qRT-PCR was done with reference to our previous articles [19 (link)]. Each slide was hybridized with 1.65μg Cy3-labeled cRNA using a Gene Expression Hybridization Kit (Agilent Technologies) and a hybridization oven (Agilent Technologies). After 17 hours of hybridization, the slides were washed with Gene Expression Wash Buffer Kit (Agilent Technologies) and scanned on Microarray Scanner (Agilent Technologies). RT-qPCR was operated by using the Applied Bio-systems 7500 Fast System (Life Technologies). The fold-change in differential expression for each gene was calculated using the comparative CT method (also known as the 2^−∆∆CT method).
The samples separated via capillary high-performance liquid chromatography were subsequently analyzed using a Triple TOF 5600þ system (AbSciex, USA). Protein identification and proteome annotation were performed using the ProteinPilotTM software package 4.5 (Applied Biosystems) and searched against the SwissProt database (March 2013) using the Mascot 2.2 search engine (Matrix Science, London, UK).

Total RNA from E. coli cells sampled at t=N− and t=N+ were extracted as above, and samples were processed by OGT (Oxford, UK) to incorporate Cy3 or Cy5 dyes and hybridized onto OGT 4 × 44 K high-density oligonucleotide arrays. Gene expression data were collected by scanning the array using an Agilent Technologies microarray scanner, and results were extracted by using Agilent Technologies image-analysis software with the local background correction option selected. Statistical analyses of the gene expression data was carried out using the statistical analysis software environment R together with packages available as part of the BioConductor project ( http://www.bioconductor.org). Data generated from the Agilent Feature Extraction software for each sample was imported into R. Replicate probes were mean summarized and quantile normalized using the preprocess Core R package. The limma R package34 was used to compute empirical Bayes moderated t-statistics to identify differentially expressed gene between time points. Generated P-values were corrected for multiple testing using the Benjamini and Hochberg false discovery rate (FDR). An FDR-corrected P-value cutoff of <0.01 was used to determine significant differential expression. Differential gene expression results only for the 60 ppGpp responsive genes are provided in Supplementary Table 1.

For analyzing the influence of acid stress on gene expression, M. tuberculosis strains (M. tuberculosis, MtbΔwhiB3, and MtbΔmshA) were grown until an A_{600 nm} of 0.3–0.4 and exposed to 7H9 medium adjusted to a range of acidic pH 4.5, 5.5, 6.2, and 7.0 and normal 7H9 (pH 6.6) for 2 h at 150 rpm at 37 °C. Total RNA from WT M. tuberculosis and MtbΔwhiB3 (pH 4.5 and 6.6) was processed and hybridized to the M. tuberculosis whole genome gene expression profiling microarray G2509F (AMADID: G2509F_034585, Agilent Technologies PLC). DNA microarrays were provided by the University of Delhi South Campus MicroArray Centre. RNA amplification, cDNA labeling, microarray hybridization, scanning, and data analysis were performed at the University of Delhi South Campus MicroArray Centre as described (16 (link)). Slides were scanned on a microarray scanner (Agilent Technologies) and analyzed using GeneSpring software. Results were analyzed in MeV with significance analysis of microarrays considered significant at p ≤ 0.05. The normalized data from the microarray gene expression experiment have been submitted to the NCBI Gene Expression Omnibus and can be queried via Gene Expression Omnibus series accession number GSE61579.

Similar to our earlier work, we used Cy-3 to label the amplified total RNA^{19 (link)}. The labeled cRNAs were purified and hybridized. Agilent Microarray Scanner was used to scan the array slides of circRNAs and mRNAs. We used Quantile algorithm and limma packages in R software to normalize the raw data. The reagents used included Low Input Quick Amp Labeling Kit, Gene Expression Hybridization Kit and Wash Buffer Kit (Santa Clara), and RNeasy mini kit (QIAGEN).

To explore the repertoire of autoantibodies in human brain tissue, we used our in-house produced planar protein microarrays. The protein array consisted of 11,520 spotted protein fragments on a glass slide, representing 10,820 unique proteins. The protein fragments were printed onto functionalized glass slides using an array jet printer (Arrayjet, Edinburgh, Scotland) as described previously^{28 (link)}. To assess tissue suitability for this method and for initial target selection, 5 randomly selected lysate patient samples were diluted 1:10 in a protein containing buffer (3% bovine serum albumin (BSA), 5% milk powder, 160 µg/ml His6ABP in 0.1% phosphate-buffered saline–Tween 20 (PBST)) and applied onto the microarray. After 2 h incubation, the slides were washed in 0.1% PBST and subsequently incubated with anti-His6ABP IgY-antibody (Immunotech HPA, Stockholm, Sweden, 1:40,000 in 0.1% PBST) at RT for 1 h. After additional washing, a secondary antibody mixture of goat anti-human IgG (a-hIgG, Alexa 647, Life Technologies, Carlsbad, CA, USA, 1:15,000 in PBST) and goat anti-chicken antibody (Alexa 555, Life Technologies, 1:15,000 in PBST) was used, and the microarray was incubated for an additional hour. After washing, scanning was performed using the Agilent Technologies Microarray scanner, and the slides were analyzed using the GenePix software.