title: GSM7820713: MxL2; Myxococcus xanthus; RNA-Seq
library_construction_protocol: Total RNA was isolated using the hot phenol method followed by ribosomal RNA (rRNA) depletion implementing previously validated protocol (Sharma 2021). Briefly, approximately 4 μg total RNA was mixed with 10× hybridization buffer (1 M Tris-HCl (pH 7.0), 2 M NaCl, RNase-free H2O), 2 μL 16S and 23S synthetic DNA oligonucleotide probes (developed by our lab previously (Sharma 2021)), and RNase-free H2O. Hybridization was performed at 45°C for 5 min. Hybridization reaction was treated with thermostable RNaseH (NEB, M0523S) and corresponding buffer, followed by incubation at 45 °C for 40 min. The reaction was transferred to ice and then treated with RNase-free DNaseI (NEB, M0303S) according to manufacturers protocol. The DNaseI treated samples were shifted to ice then followed with an Agencourt RNAClean™ XP bead cleanup (Beckman Coulter, Brea, CA) with a 1.8× volume of beads to sample as per manufacturers protocol. RNA samples depleted of rRNAs were eluted off the beads in RNase-free H2O. Libraries were generated using NEBNext® Ultra™ II Directional RNA Library Prep Kit (NEB #E7765), according to manufacturer's protocol without without PolyA selection and rRNA depletion steps.