GSM5135230: YP16_Total_KES30_rep1; Staphylococcus aureus; RNA-Seq
Bacterial cells were disrupted by cryomilling on a Retch MM400 (15 mm grinding balls, 10 ml jars) for 6 set of 3 min cycles at 15 Hz. Ref: Oh et al (2011) Cell 147: 1295-1308 Cell lysates were subjected to micrococcal S7 nuclease digestion, and  70S monosomes were collected by 10-40% sucrose density gradient. Total mRNA (alkaline hydrolysis) and the monosomal mRNAs (26-42 nt) were isolated by hot-phenol/chloroform method. The fragmented mRNAs and ribosome footprint mRNAs (RPFs) were ligated to a universal linker. Following rRNA subtraction, PAGE purification, reverse transcription, cDNA circularization, and multiplexing PCR amplification, the cDNA libraries were sequenced on Illumina HiSeq 2000