title: Next-Generation Sequencing of sgRNA spacer sequences from a CRISPRi library screen of the Salmonella enterica serovar Typhimurium 14028s genome in response to repeated monoclonal IgA treatment
design: Next-Generation Sequencing of sgRNA spacer sequences from a CRISPRi library screen of the Salmonella enterica serovar Typhimurium 14028s genome in response to repeated monoclonal IgA treatment
library_construction_protocol: Cultures of Salmonella enterica subspecies enterica serovar Typhimurium (genotype: ∆cas3::thyA) were grown to mid-log phase, washed with PBS pH 7.4, and standardized to an OD of 1.0 prior to treatment with 15 µg/mL Sal4 IgA (or left untreated) in a 16 x 125 mm test tube. Cultures were incubated for five hours at room temperature to allow the antibody to agglutinate the majority of the bacteria. After 5 h of antibody exposure, 200 µL was taken from the very top of the culture supernatant to serial dilute and plate for CFUs as well as passage the cultures overnight to repeat the growth and treatment process the following day (two total \"rounds\" of antibody treatment per sample) and enrich for the non-agglutinating subpopulation. This screening procedure was performed twice (biological replicates) each with two technical replicates.      Frozen aliquots of Salmonella enterica subspecies enterica serovar Typhimurium (genotype: ∆cas3::thyA) containing the CRISPRi sgRNA plasmid library were subcultured in Luria Bertani (LB) medium supplemented with carbenicillin at 37°C with aeration (225 rpm) in a MaxQ 4000 benchtop incubator (ThermoFisher Scientific, Waltham, MA) until mid-log phase was reached.  Optical density at 600 nm (OD600) was monitored using a GENESYS 10S UV408 Visible spectrophotometer (ThermoFisher Scientific). Cultures were then centrifuged at 6,000 x g for four minutes, washed twice in PBS pH 7.4, and standardized to an OD600 of 1.0 prior to treatment. Washed and concentrated cultures were mixed with Sal4 IgA antibodies (isolated from murine hybridoma supernatant) in a final volume of 7.5 mL and antibody concentration of 15 µg/mL in a 16 x 125 glass test tube. Cultures were treated for 5 hours at room temperature. After two rounds of antibody treatment, plasmids containing the sgRNA/spacer sequences were isolated from 3 mL of overnight culture from each sample with a QIAprep Spin Miniprep Kit (QIAGEN) according to the manufacturer's instructions. Following extraction, the concentration of purified DNA was quantified by Nanodrop (ThermoFisher Scientific).The region of the plasmid encoding the spacer sequence was amplified with a low (~10X) amplification cycle PCR program using ultramers containing Illumina barcodes and custom sample indices. The resulting PCR products were resolved on an 8% polyacrylamide gel to confirm the correct fragment length (~360 bp). The DNA pool was purified using AMPure XP reagent (at a ratio of 0.8 beads/sample volume) according to the manufacturer's instructions (Beckman Coulter, Indianapolis, IN). Final DNA concentration was quantified using a Qubit 4.0 fluorometer and dsDNA Broad-Range Assay Kit (ThermoFisher Scientific).