NextSeq 500 sequencing; Cappable-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.  ' triphosphorylated RNA was capped with 3'-desthiobiotin-TEG-guanosine 5' triphosphate (DTBGTP) (New England Biolabs) using the vaccinia capping enzyme (VCE) (New England Biolabs). Then biotinylated RNA molecules were captured using streptavidin beads and eluted with a biotin-containing buffer. RNA samples were poly(A)-tailed using poly(A) polymerase. Then the 5'-PPP or CAP structures were converted to 5'-P using CAP-Clip Acid Pyrophosphatase (Cellscript). Afterwards, an RNA adapter was ligated to the newly formed 5'-monophosphate structures. First-strand cDNA synthesis was performed using an oligo(dT)-adapter primer and M-MLV reverse transcriptase. The resulting cDNAs were PCR-amplified using a proof-reading enzyme. The libraries were amplified in 15 cycles of PCR. The generated cDNA libraries were sequenced on an Illumina NextSeq 500 system using 75 bp read length.