Miseq 2 150

P. ogarae RDP1 was isolated from the rhizosphere of pepper (Capsicum annuum) as previously described [26 (link)]. Total DNA was extracted using the Realpure Genomic DNA extraction Kit (Durviz, Spain) and sequenced by paired-end Illumina MiSeq 2×150 at Parque Científico de Madrid (Spain). Reads were quality-filtered using Trimmomatic software v0.38 [42 (link)]. The genome of P. ogarae RDP1 was assembled using SPAdes v3.14.1 software [43 (link)], read error correction and careful parameters. Prokka v1.14.6 software [44 (link)] was used to annotate the genome, using the genetic code number 11 and default parameters. The genome sequence has been deposited in the NCBI and it is publicly available under the BioProject accession number PRJNA697838.

CRISPR/Cas9 mediated FORCP knockout LS180 cells were generated by Synthego Corporation (Redwood City, CA, USA). To generate KO cells, Ribonucleoproteins containing the Cas9 protein and synthetic chemically modified sgRNA were electroporated into LS180 cells using Synthego’s optimized protocol. Editing efficiency was assessed 48 hr post-electroporation by extracting genomic DNA from a pool of transfected cells followed by PCR amplification and Sanger sequencing. The resulting chromatograms were processed using Synthego Inference of CRISPR edits software (ice.synthego.com). The pool of cells was then seeded in 96-well plates at one cell per well followed by clonal selection and RT-qPCR to determine the effect on FORCP mRNA levels.
For indel analysis using Illumina sequencing, 20 ng of gDNA was used as template to amplify around the sgRNA target site using the primers listed. Briefly, two rounds of PCR were performed using the Kapa HiFi 2X mastermix in order to generate amplicons that can be sequenced using the Illumina MiSeq 2 × 150 format. Paired-end reads were generated, merged using FLASH (Magoč and Salzberg, 2011 (link)), filtered for quality, and subsequently mapped to the reference amplicon sequence using bwa mem. Sorted and indexed BAM files, generated by samtools, were then visualized using the Integrative Genomics Viewer (IGV).

The nucleosome library (0.2 pmol/µl) is digested by MNase (0.05 U/µl) in nuclease digestion buffer (10 mM Tris–HCl, pH 8.0, 2 mM CaCl₂) for a time course of 0 (no MNase used), 5 and 10 min at 37°C. After the defined incubation time, digestion was stopped (2% SDS, 40 mM EDTA). Proteinase K (16 µg) is added to each sample, and the reaction is incubated at 55°C for 1 h. The DNA is purified from the reaction and concentrated using a Qiagen MiniElute Purification Kit. The DNA concentration of each sample is determined by the Invitrogen Quant-iT dsDNA Assay Kit and equalized. Illumina sequencing libraries were generated using NEBNext Ultra II DNA library prep kit. Individual samples are multiplexed and sequenced on an Illumina MiSeq 2 × 150. MNase-seq sequencing results are quality filtered (q > 30) and adapter trimmed using Cutadapt (26 ). The quality reads are merged and mapped to the 7500 nucleosome library sequences using Vsearch (27 (link)). The read counts and end positions are used to determine MNase protection, which is a measurement of the percentage of reads for a specific nucleosome base pair location. MNase protection is calculated for each base pair as the ratio of base pair coverage/total reads for that specific nucleosome.