Differential timing for glucose assimilation in Prochlorococcus and coexistent microbial populations at the North Pacific Subtropical Gyre

Investigation of whole genome gene expression level changes in Prochlorococcus from environmental samples. The diel gene expression analyzed in this study is further described in Muñoz-Marin, M., Duhamel, S., Björkman, K., Magasin, J., Díez, J., Karl, D.M., Garcia-Fernandez, J.M., 2021. ISME J. (to be submitted). Six probes of 60 nucleotides in length were designed for each gene, and a total of 7,501 probes (1,326 genes) were designed for Prochlorococcus. These probes were replicated 4 times in the 8 × 60K array slides, which allowed internal evaluation of signals. The sequences of all oligonucleotide probes were tested in silico for possible cross-hybridization as described below. The probe sequences were used as queries in the BLASTN against the following available nt databases in June 2017: Marine microbes, Microbial Eukaryote Transcription, and Non-redundant Nucleotides NCBI SRA website and all rRNA databases from Silva as of February 2, 2016. Agilent technology allows 5% nt mismatch in the whole probe region; thus, sequences with a range of 95% to 100% nt identity to the target probe are detected. Therefore, all probes with BLASTN hits with ≥95% over 100% of the nt length were deleted. Next, the probe sequences that passed the cross-hybridization filter were clustered using CD-HIT-EST at 95% nt similarity to select unique probes for Prochlorococcus. In addition, standard control probes (IS-62976-8-V2_60Kby8_GX_EQC_201000210 with ERCC control probes added) were included randomly as part of the Agilent Technology array to feature locations on the microarray slide. The final design of the microarray was synthesized on a platforms of ca. 62,976 experimental probes and 1,319 control probes on each 8 × 60K array slide.