GSM7506306: Library, replicate 2; Bacteroides thetaiotaomicron; OTHER
Libraries were prepared using QIAseq Ultralow Input Library Kit according to manufacturer instructions. 10ng input DNA were used and 8 PCR cycles were performed. Libraries were quantified by QubitTM dsDNA HS Assay Kit (3.0 Fluometer; ThermoFisher) and quality was checked using 2100 Bioanalyzer with High Sensitivity DNA kit (Agilent Technologies) before pooling. Sequencing of pooled libraries, spiked with 50% PhiX control library, was performed in paired-end mode with 150 nt read length on the NextSeq 2000 platform (Illumina) with a P1 Kit and 20 Mio reads per library. Demultiplexed FASTQ files were generated with bcl2fastq2 v2.20.0.422 (Illumina). We incubated the culture at 37°C until it reached the indicated OD, at which point we took a 2 mL aliquot, pelleted it, discarded the supernatant, and stored the bacterial samples at -20°C until further processing.  Bacterial pellets were resuspended in 100 μL of PBS containing 250 μg lysozyme, mixed with 100 μL lysis buffer (100 mM Tris-HCl pH 8.5, 200 mM NaCl, 0.2% SDS, 5 mM EDTA), and lysed by incu-bating at 37°C for 5 min. Proteins were then degraded by addition of 1 μL of Proteinase K (NEB #P8107) and incubation at 56°C for 30 min, following which RNA was digested with 0.5 μL of RNase A (Thermo #EN0531) and incubation at 56°C for 5 min. The remaining gDNA was purified from each sample via phenol-chloroform extraction, followed by ethanol precipitation.  The genomic regions coding for gRNAs and arrays were PCR-amplified in a 50 µL reaction con-taining 100 ng gDNA with oligonucleotides AWO-577/AWO-578 (annealing temperature: 55°C) and AWO-575/AWO-576 (annealing temperature: 63°C).