Instrument: Illumina HiSeq 2000
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: SINGLE
Construction protocol: Bacterial cells were disrupted by cryomilling on a Retch MM400 (15 mm grinding balls, 10 ml jars) for 8 set of 3 min cycles at 15 Hz. Ref: Oh et al (2011) Cell 147: 1295-1308 Cell lysates were subjected to micrococcal nuclease digestion, and monosomes were collected by 10-55% sucrose density gradient. Total mRNA and the monosomal mRNAs were isolated by hot-phenol/chloroform method. Total mRNAs were fragmented by alkaline hydrolysis. The fragmented mRNAs and ribosome footprint mRNA (RPF) 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