After hybridization, slides were washed for 5 min at room temperature with CGH Wash Buffer 1 and 1 min at 37°C with CGH Wash buffer 2 (Agilent Saracatinib clinical trial Technologies) and scanned immediately, using an Agilent High Resolution Microarray Scanner, at 5 micron resolution, 100% PMT. Scanned images were quantified using Feature Extraction software v 10.7.3.1 (Agilent Technologies) and statistical
analysis of raw intensity data was performed in GeneSpring v12.1 (Agilent Technologies). Data for 3 independent biological replicate experiments were analysed. Data for each sample were normalized using a 75th percentile shift plus baseline normalized to the median of the related control sample for each biological replicate. Statistically
significant (p < 0.05) differences of more than 10-fold between the two strains were identified in an unpaired t-test with Benjamini and Hochberg false ABT-263 order discovery rate correction. Real time RT-PCR Bacteria were cultured as described for EM in CDM, 5.5 mM AZD2014 order glucose, pH 7 to mid-log phase. Double volume of RNAprotect® bacteria reagent (Qiagen) was added. RNA was isolated and real-time RT- PCR performed as described previously to analyze expression of the first gene of the capsule operon, cpsA and competence gene comX [61]. The primer sequences for comX were: forward primer, 5′-TGT ATG AAG AAG TCC AAG GGA CTG T-3′, reverse primer, 5′-GTA AGC AGA GCA TGC CTT CTT G-3′ and probe 6-FAM-CCC ATA AAT GAA GGT AAT ATT-MGB_NFQ. Benzatropine Statistical analysis GraphPad Prism v6.03 software for Windows (www.graphpad.com) was used to perform the statistical analysis with the unpaired t test with Welch’s correction. p ≤ 0.05, two-tailed, was considered significant. Nucleotide sequence accession numbers The genome raw sequences of the Illumina runs are deposited in the short reads archive (http://www.ncbi.nlm.nih.gov/sra): 307.14, encapsulated (SRX485275) and 307.14, nonencapsulated (SRX485278). BioProject accession PRJNA241072 (http://www.ncbi.nlm.nih.gov/bioproject/241072). Results Clinical pneumococcal isolate 307.14 consists of two variants: one encapsulated and
one nonencapsulated On plating the nasopharyngeal isolate 307.14 onto CSBA plates, a mixture of large and small colonies was observed. The large colonies made up approximately 50% of the total and were serotyped as 18C (hereafter referred to as 307.14 encapsulated) whereas repeated attempts to type the small colonies led to the conclusion that they were nonencapsulated (hereafter referred to as 307.14 nonencapsulated). RFLP was performed on both phenotypes and confirmed that they were of the same genetic background (RFLP type 14, data not shown). Following subculture in CDM with 5.5 mM glucose, the bacteria were exposed to FITC-dextran. Figure 1A shows that by fluorescence microscopy two distinct sizes of bacteria could be observed.