GSM5776716: KrrA-FLAG+205#1; Bacillus anthracis; RIP-Seq
The cell lysates (supernatants) were collected after centrifugation. Aliquots of 1% lysates were diluted with buffer A and kept at −80°C to serve as the input control (1% of input RNA). The remaining lysates were incubated with α-FLAG M2 magnetic agarose beads (Sigma, Cat# M8823) on a rotating wheel for 2 h at 4°C for immunoprecipitation. The bead slurry was recovered using a magnetic stand, washed twice with 1ml of high-salt wash buffer (50mM Tris pH7.4, 1 M NaCl, and 1mM EDTA), then washed once with 1 ml of IP wash buffer (50mM Tris pH7.4, 10mM MgCl2, and 0.2% Tween-20). Aliquots of 5% beads were saved for immunoblotting to examine the immunoprecipitation efficiency. The remaining beads were subjected to DNAase treatment and the protein–RNA complexes were eluted with 3X FLAG peptide according to the manufacturer's protocol. All samples including 1% input RNA samples were incubated in buffer B (50mM Tris pH7.4, 10mM DTT, 1% SDS, 1 mg ml-1 protease K, and 5mM EDTA) at 70oC for 45 min to reverse crosslinking. Enriched RNAs were extracted using Trizol, precipitated using isopropyl alcohol with linear acrylamide, washed twice with 75% ethanol, and quantified using a NanoDrop spectrophotometer. Library construction was performed using NEBNext Ultra II RNA Library Prep Kit for Illumina and NEBNext Multiplex Oligos for Illumina