title: RNA-Seq of myxococcus xanthus: 24h submerged culture monolayer
design: Briefly, 2-3 g of RNA was mixed in hybridization buffer (0.1 M Tris-HCl [pH 7.0], 0.2 M NaCl) with 76 DNA oligonucleotides (0.17 M/oligo) that anneal to M. xanthus 16S and 23S rRNAs, the mixture was incubated in a thermocycler at 95C for 2 min, then ramped down to 45C at a rate of 0.1C/s, followed by additional incubation at 45C for 5 min.  The annealed samples were treated with Hybridase Thermostable RNase H (Epicentre) in order to hydrolyze RNA-DNA hybrids and digested with DNase I (Roche) to remove remaining oligos.  Agencourt RNAClean XP beads (Beckman Coulter) were used for cleanup of the rRNA-depleted RNA samples.  qPCR verification was performed to confirm successful (i.e., ~90%) rRNA depletion.  The KAPA RNA HyperPrep kit (KAPA Biosystems) was used as described in the product manual to generate adapter-ligated libraries for multiplex sequencing.  The Qubit 1X dsDNA HS (High-Sensitivity) Assay Kit (ThermoFisher Scientific) was used to measure library concentration.