5 protocols
Arrays were scanned using an Agilent C scanner at 5um resolution (20 bit scan).
Labeled RNA samples were normalized at 700ng of each dye component, fragmented and hybridized to a custom anti-sense probe 2x11K microarray (AMADID 015123) using Agilent buffers.
Pooled total RNA samples were directly labeled (no amplification) with Kreatech ULS aRNA Labeling Kit.
WT and delta P3 cells from plates were grown in liquid BG-11 for 4 days in PA LL conditions. They were re-suspended to about 1x107 cells ml-1 in 100 ml of BG-11 with 25 mM Hepes-NaOH pH 7.5, then grown for 1.25 days in photoautotrophic conditions till the cell count reached around the mid-log level. 5 mM glucose was then added and the cultures were grown for another 2 days in 12LD cycles. Samples for RNA extraction were taken after adding glucose at 1 h in day 1 in the light (L1) and in the dark (D1), and 1h in day 2 in the light (L13), and in the dark (D13). Two biological replicates and four technical replicates were used per time point per culture including a dye swap.
Cells from the four time points were spun down and the pellet was frozen in STET buffer at -80?C. RNA was extracted using Tri-reagent (Ambion), Dnase-treated, and column purified (Zymo research).