DNA-SEQ of synthetic library for quantifyingribosome density on transcript: regular, steady-state growth condition, polysome fraction #4
The synthetic E. coli library comprising 244,000 variants of a fluorescent reporter was grown to steady state in rich MOPS medium. Culure were fast filtered and flash frozen in liquid nitrogen. Cells were mechanically lysed while frozen. The cell lysate was ultracentriguged on a sucrose gradient to separate the polysome fractions. The five first fractions were recovered separately. RNA was then extracted. Reporter trancripts were reverse transcribed in a targeted fashion. The variable region of the reporter was then PCR amplified. Primers used for generating the sequencing libraries contained handles for sequencing as well as custom sequencing barcodes for multiplexing. Libraries from different fractions were pooled with other libraries for sequencing (150PE). Resulting reads were demultiplexed through identification of the custom barcodes, which was then trimmed from the sequences along with the priming region. The fastq files contain these trimmed, demultiplexed reads. Reads with no assignable barcode were thrown away. No other data processing was performed.