Caliper labchip gx

cDNA from each sample was generated from 10ng of total RNA using SMARTer Ultra Low Input RNA kit v4 (Clontech). The cDNA was amplified by 8 PCR cycles, followed by QC analysis on BioAnalyzer 2100 (Agilent). Sequence libraries were produced from 150pg of cDNA using Nextera XT DNA library kit (Illumina), cleaned up with AMPure XP beads, and QC checked with Caliper LabChip GX. Single-end sequencing data were generated on an Illumina HiSeq 2500, at a depth of 30 million reads per sample, with read length 50bp.

Samples of 4 l were filtered using 0.22 μm Pall Supor filters (Pall Corporation, Port Washington, NY, USA). DNA extraction from filter was carried out using the RapidWater DNA extraction kit (MoBio, Carlsbad, CA, USA). Sequencing libraries were prepared using Nextera XT DNA Library Preparation Kit (Illumina, San Diego, CA, USA) following the manufacturer instructions, and quality checked using Caliper LabChip GX. Sequencing was performed on an Illumina HiSeq 2500 platform (Illumina, San Diego, CA, USA) with 100 bp paired-ends and an insert size of 250 bp. Raw sequencing reads were quality checked using fastq-mcf [45 ], and reads derived from human contamination were removed after mapping on the human genome (version hg19) using bowtie2 [46 (link)]. Adaptors and residual synthetic constructs were removed first mapping the reads on the genome sequence of the phage Phy174, and then using trim galore [47 ].
Microbiome profiling based on a database of taxon-specific marker genes was performed using MetaPhlAn2 [48 (link)]. The output of MetaPhlAn2 analyses was plotted using GraPhlAn [49 (link)].

Gene deletions were accomplished using our previously established CRISPR/Cas9‐based genome editing protocol (Zerbini et al., 2017). The preparation of the 90 pCRISPR‐SacB‐gDNA plasmids were done using double‐stranded synthetic oligonucleotides (Merck, Darmstadt, Germany) encoding for the guide RNA reported in Table S4. Stepwise single gene mutagenesis was achieved by co‐transformation of electrocompetent cells of the recipient strain (E. coli BL21(DE3)ΔompA_pCasRed, E. coli Δ01_pCasRed, etc.) with the gene‐specific pCRISPR‐SacB‐gDNA and its respective double‐stranded donor DNA (ds‐dDNA) (Table S5). Gene‐specific primer pairs used for screening of colonies with GoTaq Green Master Mix (Promega, Madison, WI USA) are listed in Table S6. Genomic DNA was extracted from E. coli BL21(DE3)ΔompA and E. coli BL21(DE3)Δ58 using PowerSoil DNA Isolation Kit (Mo Bio Laboratories, Carlsbad, CA USA) and the genomes were fully sequenced. Briefly, libraries were prepared using the Nextera XT DNA Library Preparation Kit (Illumina, San Diego, CA USA), and library quality was assessed using the Caliper LabChip GX (High‐Throughput Bioanalyzer) following manufacturers guidelines. Sequencing was performed on the HiSeq 2500 system (Illumina, San Diego, CA USA).