GSM8465248: 4exo_rendseq_pooled; Bacillus subtilis; RNA-Seq
Prior to RNA extraction, cell pellets were washed with 10-15 mL ice cold 10 mM Tris pH 7.0 to remove contaminating nucleic acids that precipitate from 2xYT in methanol. For Rend-seq, RNA was extracted from cell pellets using either an RNeasy Mini Kit (Qiagen) with on-column DNase treatment, an RNeasy Plus Mini Kit (Qiagen) or through RNAsnap extraction (Stead et al., 2012) followed by Turbo DNase treatment. Following RNA extraction and gDNA depletion, 20 µg of purified RNA was treated with MICROBExpress Bacterial mRNA Enrichment Kit (ThermoFisher) per the manufacturer's instructions and precipitated with isopropanol. Libraries were constructed as per the Rend-seq protocol described in Lalanne et al., Cell 2018.