RNA-Seq of sgRNA pool targeting Citrobacter rodentium
A 236 kb fragment of the Citrobacter rodentium DBS100 genome was screened for 5-NGG-3 PAM sequences and 31,596 sites were identified. We took the 20 bp upstream and ordered these alongside 200 non-targeting sequences as a pool from Twist Bioscience after appending the sequence 5-CCTGGTTCTTGGTCTCTCACG-3 upstream of the sgRNA and 5-GTTTTAGAGACCGCTGCCAGTTCATTTCTTAGGG-3 downstream to allow for efficient and directional cloning. The sgRNA pool was made double stranded by PCR amplification with primers DE5231 and DE5224 (Supplementary Table S11) using 1 ng of single stranded sgRNA template and cloned into pTevSpCas9 plasmid as described previously. Cloning reactions were electroportated into E. coli EPI300 in 5 independent reactions and all added 500 mL of LB (10 g/L tryptone, 5 g/L yeast extract, 10 g/L sodium chloride) supplemented with 25 mg/mL chloramphenicol and 0.2% w/v D-glucose to grow at 37C with shaking at 225 rpm overnight. The plasmid pool was then isolated using the Monarch Plasmid Miniprep Kit (NEB) according to manufacturers specifications. In 10 independent reactions, 50 ng of the sgRNA pool was electroporated into 100L C. rodemtium competent cells and allowed to recover in 1 mL of non-selective 2xYT media (16 g/L, 10 g/L yeast extract, and 5 g/L NaCl) for 30 minutes at 37C with shaking at 225 rpm. The recovery was then split and 500 L was added to 500 L of inducing 2xYT (0.4%) (w/v) L-arabinose and 50 mg/mL chloramphenicol) or to 500 L of repressive 2xYT (0.4%) (w/v) D-glucose and 50 mg/mL chloramphenicol) and incubated for 90 min at 37C with shaking at 225 rpm. The resulting cultures were then added to 50 mL of LB (10 g/L tryptone, 5 g/L yeast extract, 10 g/L sodium chloride) supplemented with 25 mg/mL chloramphenicol and 0.2% w/v D-glucose and grown overnight at 37C with shaking at 225 rpm. Plasmids were then isolated using the Monarch Plasmid Miniprep Kit (NEB) according to manufacturers specifications. The sgRNA locus was then PCR amplified using primers (Supplementary Table S11) containing Ilumina adapter sequence, 4 random nucleotides, 12-mer barcodes to specify the replicate, and plasmid-specific nucleotides at the 3 end. The resulting amplicons were sent for 150 bp paired-end Illumina NextSeq High Output sequencing the London Regional Genomics Center (London, ON).