GSM5114322: [Cappable-seq] B. anthracis 34F2 WT #3 unenriched; Bacillus anthracis; OTHER
[ChIP-seq] Cells were pelleted and treated with 1% formaldehyde for for crosslinking. 250 mM glycine was added for quenching. After pelleting and resuspension in lysis buffer with cOmplete Mini Protease Inhibitor Cocktail, the samples were sonicated using Covaris S2. Supernatants were combined with Anti-FLAG M2 affinity gel for enrichment, followed by wash and protease K treatment at 65°C. [Cappable-seq] RNA was extracted using TRIzol after incubating the pellet with 2% SDS. After removing DNA with DNA-free DNA removal kit, RNA samples followed the protocol described before (Ettwiller et al, BMC Genomics 17:199, 2016) [3´-RACE] RNA was extracted using TRIzol after incubating the pellet with 2% SDS. DNA was removed with DNA-free DNA removal kit. [RNA-seq] RNA was extracted using TRIzol after incubating the pellet with 2% SDS. DNA was removed with DNA-free DNA removal kit. rRNA was depleted by the protocol described before (Culviner et al, mBio, 2020), followed by fragmentation with the treatment with a divalent cation and heat. [ChIP-seq] Library was constructed with NEBNext Ultra II DNA Library Prep Kit for Illumina. [Cappable-seq, 3´-RACE, and RNA-seq] Library was constructed with NEBNext Multiplex Small RNA Library Prep Set for Illumina.