title: GSM6203286: CRISPRi-seq, mock, rep1-4; Streptococcus salivarius; OTHER
library_construction_protocol: After genomic extraction (GenElute, Sigma Aldrich) from at least 1.5 × 109 CFU per replicate, the locus containing the gRNAs was PCR-amplified prior to their deep sequencing. An optimized PCR protocol was used with high primer concentration (5 µM), low template genomic DNA (2 ng/µl), and low number of cycles (15 cycles) to avoid any chimeric products due to the highly randomized gRNA sequences. The 219-pb amplicons were next gel-purified (Monarch DNA Gel Extraction kit, NEB) and sent with a minimum amount of 4 pmol for Illumina sequencing. High-Seq Illumina sequencing was performed with 30% Phix and generated an average of 30 M reads per replicate. The gRNA library was designed by selecting all the 20 nt followed by a PAM sequence within the genome of HSISS4. The library was filtered to keep only gRNAs targeting the coding strand in CDSs and targeting both strands in intergenic regions. We ended up with a high-density library of 83,104 gRNAs (Table S1A, random_gRNA_HSISS4). We ordered the gRNAs as single strand DNA (Twist BioSciences) and amplified the oligo-pool thanks to common upstream and downstream region using primers AK475 (CGGACTAGCCTTATTTAAAC) and AK476 (GTCCCCCTGGTATAATAACTATA). To reduce any amplification sequence-bias, we used 10 cycles of amplification. The PCR products were then purified (Monarch kit, NEB) and Gibson-joined to the pre-amplified upstream homologous region of the neutral locus gor (downstream of HSISS4_00325) containing an erythromycin resistance gene and to the downstream homologous region of the same locus fused to a P3 constitutive promoter. We performed 20 independent Gibson assemblies, which were latter transformed by natural transformation into 20 independent cultures of HSISS4 containing at least a lactose-inducible dCas9 module (PF6-lacI; Plac-dcas9). For every library produced, we calculated the number of transformants to obtain at least 15-fold transformants over the diversity rate, ensuring theoretically that 99,9% of the diversity would be present in the library. We finally pooled all the transformants in PBS and measured OD600 prior to storage at -80°C.