GSM8465261: 4exo_ylbF_3exo_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. All 3′ end sequencing samples were purified through RNAsnap. In the RNAsnap protocol, cells were resuspended in 500 µL extraction solution (95% Formamide, 1% BME, 0.025% SDS, 18 mM EDTA) and transferred to a 2 mL tube containing 200 µL chilled 0.1 mm diameter zirconia beads. These tubes were vortexed for 10 minutes using a horizontal vortex adapter and subsequently incubated at 95 °C for 7 minutes. Samples were spun at 15,000 g, after which 400 µL was removed and diluted 1:5 with 1.6 mL DEPC-treated water. This mixture was precipitated with isopropanol and resuspended in 10 mM Tris, pH 7.0. At least 30 µg of RNA was then treated for 30 minutes at 37°C with 10 µL Turbo DNase in a 100 µL reaction. 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. 3′ end sequencing libraries were prepared in a manner similar to Rend-seq (Lalanne et al., Cell 2018), with deviation in the order of reactions (for full protocol, see Herzel et al., NAR 2022). Prior to fragmentation, 1.8 µg of rRNA depleted RNA was ligated to an adenylated adapter as described for Rend-seq. This ligation product was then fragmented and RNA species of sizes 33-63 nt were purified via denaturing polyacrylamide gel extraction. From this point onwards, the library preparation is identical to Rend-seq (Lalanne et al., 2018).