Illumina HiSeq 3000 sequencing; RNA-seq analysis of Myxococcus xanthus at various developmental time points
Exponentially growing M. xanthus in CTT were harvested at 5,000 g for 5 min and resuspended in MC7 buffer (10 mM MOPS pH 6.8, 1 mM CaCl2) to 7×109 cells ml-1. 1 ml of concentrated cells was added to 10 ml of MC7 buffer in a polystyrene Petri dish with a diameter of 9.2 cm (Sarstedt). Total RNA from M. xanthus cells developed under submerged conditions was extracted from cells using TRI Reagent (Sigma-Aldrich) according to the manufacturer's protocol. Purified RNA was treated with TURBO DNA-free™ Kit (Invitrogen) according to the manufacturer's protocol. RNA integrity was analyzed by 1% agarose gel electrophoresis. rRNA depletion was conducted with 1 µg total RNA using Ribo-Zero rRNA Removal Kit Bacteria (Illumina), followed by library preparation with NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England Biolabs). Library preparation included 11 cycles of PCR amplification. Quality and quantity were assessed at all steps via capillary electrophoresis (TapeStation, Agilent Technologies) and fluorometry (Qubit, Thermo Fisher Scientific).