RNA-SEQ of synthetic library for quantifying mRNA decay: 2.5 min after transcription arrest, replicate #1
The synthetic E. coli library comprising 244,000 variants of a fluorescent reporter was grown to steady state in rich MOPS medium. Sample was taken 2.5 min after arresting transcription with antibiotic treatment. Cells were incubated with RNAprotect, peleted and lysed. A fixed volume of lysate from a culture containing a mix of 8 standard reference sequence was spiked into the solution. RNA was then extracted and ribosomal RNA substracted using RiboZero. Reporter trancript 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. This library was pooled with other libraries similarly generated at other time points 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.