GSM7947760: RNA-seq_3KO 1h post sporulation induction, replicate 1; Bacillus subtilis; RNA-Seq
RNA was isolated from sporulating WT and 3KO strains. Cultures were harvested hourly for seven hours into sporulation, beginning at T0 (prior to sporulation induction), resulting in a total of eight timepoints. Before harvesting, cultures were treated with 0.3 mM chloramphenicol. Cells were collected by filtration and flash frozen in liquid nitrogen. Purification of mRNA, ribosomal footprint isolation and library preparation was performed as described earlier. Briefly, frozen pellets were ground using mortar and pestle with the addition of aluminum oxide and lysis buffer. The extracted total RNA was used for purification of mRNA and ribosomal footprint isolation. rRNA depletion was performed to yield mRNA which was then fragmented by alkaline hydrolysis. Ribosomal footprint isolation was performed by MNase digestion of the total RNA followed by size selection using polyacrylamide gel electrophoresis. The obtained mRNA fragments and ribosomal footprints were end-repaired (dephosphorylation of 3'ends followed by phosphorylation of 5' ends) and used for preparation of cDNA sequencing libraries with NEBNext Multiplex Small RNA Library Prep Set for Illumina, according to the manufacturer's guidelines. Sequencing was performed on an Illumina NextSeq500 by Genomed S.A. (Warsaw, Poland) and Genome Facility at CeNT (Warsaw, Poland); single-end 50bp reads were sequenced.