title: GSM5419895: BS168-yerA-KO-1; Bacillus subtilis subsp. subtilis str. 168; RNA-Seq
library_construction_protocol: The cell pellets were resuspended by 1 mL RNAlater (ThermoFisher Scientific, USA, LOT. AM7020) and flash frozen by liquid nitrogen, and stored at −80 °C until use. Upon RNA extraction, the RNAlater were removed by centrifugation and the cell pellet were dissolved in 0.2 mL buffer lysis (30 mM Tris pH 6.2, 10 mM EDTA, 0.1 mg/mL lysozyme, DEPC-treated water), then incubation at 37 °C for 30 min. Subsequently, 1 mL TRIzol reagent (ThermoFisher Scientiﬁc Inc., USA) was added and mixed well, following mixed with 0.2 mL chloroform. The mixture was centrifuged at 15000 ×g for 15 min at 4 °C, and the aqueous phase was transferred to a different RNase-free tube. Then 0.3 mL of saline solution (0.6 M NaCl, 0.4 M sodium citrate), and 1 mL of isopropyl ethanol were added, and the mixture was stored at −20 °C for 2 h. After the mixture was centrifuged at 15000 × g for 20 min at 4 °C, the pellet containing the total-RNA was washed twice with 1 mL of 70% (v/v) ice-cold ethanol. Finally, the RNA pellet was air dried and dissolved with 200 µL of RNase-free water. The concentration and quality of RNA was analysed by NanoDrop 2000 ONEc (thermofisher scientific, USA).