Tape station bioanalyzer

Libraries for BCR sequencing were prepared as previously described [10 (link)]. All libraries were checked for quality and quantity using the Tapestation Bioanalyzer (Agilent, Santa Clara, CA, USA) and the Qubit™ dsDNA HS Assay Kit (Thermo Fisher, Waltham, MA, USA) on a Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA, USA). Sequencing was performed on a MiSeq instrument (Illumina, San Diego, CA, USA) using 300 bp paired-end reads. For extraction of the CDR3 amino acid sequences of IGHV, bioinformatics analysis was performed using the MiXCR software (version 3.0.13, https://github.com/milaboratory/mixcr, accessed on 26 May 2021) [12 (link)] standard pipeline with export of only productive IGHV clones. For further analysis, a cut-off at 0.01% of total sequencing reads was applied. In addition, whole sequences were extracted into fasta format for analysis of the IGHV mutation status via the IMGT/V-QUEST tool [13 (link)]. BCR sequencing data are deposited in the Sequence Read Archive, NCBI, NIH (BioProject: PRJNA475208; SRA accession code SRP150049 and BioProject: PRJNA725403).

DNA libraries for each sample were prepared from isolated chromatin using the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB) and NEBNext Multiplex Oligos for Illumina (NEB) according to the manufacturer’s instructions. No size selection was performed after adaptor ligation. DNA was amplified by PCR with 11 amplification cycles. Library concentration and fragment size distribution were checked using D1000 ScreenTape with TapeStation bioanalyzer (Agilent). Library quantitation was done with the NEBNext Library Quant Kit for Illumina (NEB) according to the manufacturer’s instructions. Pooled libraries were sequenced on an Illumina NextSeq 500 with 75 bp SE reads.

Bacterial cells were collected from mono- and co-cultures after 6 h of growth at 28°C in a partial stigma-mimicking medium. RNA was extracted using the RNeasy PowerLyzer Tissue & Cells Kit (Qiagen) according to the manufacturer’s instructions. The RNA was eluted in 50 µL nuclease-free water. Quantity and quality control of the RNA was assessed by using Qubit RNA Broad-Range Assay (Invitrogen) and with RNA ScreenTape analysis on Agilent TapeStation Bioanalyzer. Before Illumina library preparation, rRNA depletion was conducted using the NEBNext rRNA Depletion Kit (Bacteria, New England Biolabs) according to the manufacturer’s recommended protocol. Then, DNA removal coupled with cDNA synthesis was conducted using NEBNext Ultra II RNA Library Prep Kit for Illumina (New England Biolabs) according to the manufacturer’s recommended protocol. DNA quantity was checked using the Qubit dsDNA HS Assay Kit (Invitrogen). Library sequencing was conducted on an Illumina NovaSeq platform through services provided by the Yale Center for Genome Analysis.

The quality of barcoded libraries was determined by electrophoresis using TapeStation Bioanalyzer (Agilent, Santa Clara, CA). Libraries were quantified using QIAGEN QuantArray kit and adjusted to 2 nM, then pooled in equal volume. The pooled amplicons were diluted to 12 pM and sequenced using Illumina MiSeq and the protocol for paired-end (2 × 276 bp) sequencing V3 kit (Illumina, San Diego, CA).
Since the main focus of this study was to characterize the mycobiome, amplicon 16S rRNA sequences were not discussed. ITS sequences obtained from each PMA treated sample were compared with the UNITE database (version 8.1, released 2019-02-02) (Nilsson et al., 2019b (link)) using NCBI BLASTn (version 2.6.0+, parameters: max_target_seqs 5, word_size 5, e-value 1e-5) (Altschul et al., 1990 (link)). The top hit to the database was retained for each sequence and a table containing the abundance of each ITS amplicon sequence types (STs) in each sample was generated. Canonical correspondence analysis (CCA) (Braak, 1986 (link); Legendre and Legendre, 2020 ) from the Vegan R package (Oksanen et al., 2013 ) was used to analyze the distribution of samples with respect to their ST constituents. The variation in distribution among STs was analyzed using the sampling event as a linear predictor. The effect of variation present in control samples was removed prior to analysis.