deep sequencing of DNA amplicons from plasmids inside cell library
An entire cryostock of cell library was resuspended in 100 mL of LB media supplemented with 50 ug/mL Chloramphenicol (Cm) in a 1 L Erlenmeyer flask and incubated at 37 C, 300 RPM agitation for 12 hours. Afterwards, it was diluted to an OD600 of 0.05 in 50 mL EZRich media (Teknova) supplemented with 50 ug/mL Chloramphenicol (Cm) in a 1 L Erlenmeyer flask and incubated at 37 C, 300 RPM agitation for 2 hours until the OD600 reached 0.3. Again, the culture was diluted to an OD600 of 0.05 in 250 mL EZRich media supplemented with 50 ug/mL Chloramphenicol (Cm) in a 1 L Erlenmeyer flask and incubated at 37 C, 300 RPM agitation for 1.5 hours until the OD600 reached 0.25 and the cells were harvested. All characterization steps were done in triplicate. Three 50 mL aliquots were taken and used to perform a plasmid extraction using the plasmid DNA mini kit (Omega Biotek). Three 4 mL samples of the culture were taken and mixed with 8 mL of RNAprotect (Qiagen) to fix cells and stop RNA degradation. These samples were processed to determine the cell library composition and mRNA levels at the T0 timepoint. Rifampicin was then added to the remaining bacterial culture to a final concentration of 500 ng/uL to halt transcriptional initiation. Three 4 mL samples were each taken at 2, 4, 8, and 16 minutes after the addition of rifampicin, followed by mixing with 8 mL RNAprotect. Fixed cultures were then pelleted at 5000 x g for 10 minutes, followed by RNA extraction (Total RNA Purification, Norgen Biotek) and removal of contaminant DNA (Turbo DNase, Ambion). Spike-in control RNA was produced using HiScribe T7 High Yield RNA Synthesis (NEB) from a linear DNA template that uses a T7 promoter to produce mRNA of similar size to the designed mRNAs with identical sites for cDNA synthesis and PCR amplification of the barcode region, though using a distinct barcode sequence. The concentrations of the extracted total RNA from the cell library and the spike-in control RNA were measured with the Quant-iT RNA assay (Thermo Fisher). Spike-in control RNA and total RNA were combined at the ratio of 10 attomoles of spike-in control to 1 ug of total RNA. Samples were then depleted of rRNA (NEBNext rRNA depletion, NEB), followed by cDNA synthesis (SuperScript IV first-strand synthesis, Invitrogen) using a reverse transcript-specific primer that binds upstream of the barcode region. Barcoded amplicon library generation for DNA-Seq and RNA-Seq were then carried out via PCR amplification of the barcode region of the either the purified plasmid DNA or the purified cDNA, respectively, using 25 cycles and gel extraction. DNA amplicon libraries were then submitted for deep sequencing, which includes adapter library preparation and flow cell loading (Illumina NovaSeq, Genewiz). Read counts were determined from FASTQ files by mapping and counting barcode sequences, employing a hash look-up table and multi-threaded operation. All barcode sequences were designed to have pairwise Hamming distances greater than 2, enabling unique barcode mapping with at most one nucleotide mutation.