Solid 5500xl system

Glomeruli of nephrin::CFP mice were isolated with magnetic dynabeads as previously described [37 (link)]. Afterwards, isolated glomeruli were cultured on collagen IV-coated μ-slides (ibidi GmbH, Munich, Germany) in phenol red-free RPMI 1640 medium (Lonza Group Ltd., Basel, Switzerland) supplemented with 10% FBS (Thermo Fisher Scientific), 100 U/mL penicillin, and 0.1 mg/mL streptomycin (Life technologies). Glomeruli were treated with Doxorubicin (50 µM, Sigma-Aldrich, St. Louis, MI, USA). After 3 days, RNA samples from Doxorubicin-treated and untreated control glomeruli were prepared for sequencing on a 5500xl SOLiD™ system (Life Technologies, Carlsbad, CA, USA) using recommended protocols as described previously [38 (link)]. Subsequently, the samples were normalized using DeSeq2 (Bioconductor). For the evaluation of statistical significance, a q-value was calculated using the Wald test, followed by a Benjamini-Hochberg multiple test correction. The result was considered statistically significant when the q value was below 0.05.

The genomic DNA of C. clavata BAUA-2787 was extracted using a DNeasy Plant Mini Kit (QIAGEN, Manchester, UK) after 2 days of cultivation in 100 mL CM medium at 26°C and 130 rpm. The DNA libraries were prepared with a 5500 SOLiD Mate-Paired Library Kit and sequenced by a 5500xl SOLiD system (Life Technologies, Carlsbad, CA, USA). A Nextera DNA Sample Prep Kit was also used for the library preparation, and the libraries were sequenced using the MiSeq platform (Illumina, San Diego, CA, USA). The hybrid de novo genome assembly (Ikegami et al., 2015 (link)) was performed using the MiSeq and SOLiD short read data. Additionally, the protein-coding genes in the draft genome were predicted by a combination of the gene prediction programs, ALN (Gotoh, 2000 (link)) and GlimmerHMM (Majoros et al., 2004 (link)). NCBI BLAST (http://blast.ncbi.nlm.nih.gov/) and the Pfam protein family database (http://pfam.xfam.org/) were used for sequence homology and conserved domain searches, as appropriate.

DNA fragment libraries were prepared according to the manufacturer's protocol (Fragment Library Preparation, Publication Part Number 4460960; Life Technologies, Tokyo, Japan). Briefly, 3 μg genomic DNA was sheared, end‐repaired, and ligated with primer adaptors. After ligation, PCR amplification (six cycles) was performed to enrich the targeted sequences. The average fragment size of the DNA library was verified by Bioanalyzer (Agilent Technologies, Santa Clara, CA). The fragmented DNA was hybridized using a TargetSeq Exome Enrichment System (Life Technologies). After washing, the captured DNA libraries were amplified using 10 PCR cycles. P1 beads were prepared using a SOLiD EZ Bead Emulsifier (Life Technologies); the emulsion PCR was carried out in a SOLiD EZ Bead Amplifier (Life Technologies) using the E80 setting. The beads carrying the amplified template DNA were purified on a SOLiD EZ Bead Enricher (Life Technologies), and the purified beads were loaded onto a SOLiD 6‐lane FlowChip (Life Technologies) and incubated for 1 hour at 37°C. Then, the beads were sequenced using a 5500xl SOLiD System (Life Technologies).

WES was performed on genomic DNA extracted from peripheral blood cells of all patients. DNA extraction was undertaken using the CHEMAGEN robot (Chemagen Biopolymer-Technologie AG, Baesweiler, Germany) and the sequencing was performed using the SureSelect Human All Exon kit V5 for library preparation (Agilent Technologies, Santa Clara, CA, USA) and ran on a 5500xl SOLiD™ system (Thermo Fisher Scientific, Massachusetts, USA). The sequencing reads were aligned to the hg19 reference genome using the software provided by the sequencer. Variant calling was performed using both the Lifescope and GATK 3.0 suites. For LifeScope, variant QC was performed employing the diBayes parameter of low astringency level (dibayes.het.min.start.pos = 2 and dibayes.hom.min.nonref.start.pos = 2). Additionally, we removed variants with a depth < 30X, variants with a calculated strand bias p value > 0.05, and all alternative allele variants with < 4 reads to minimise artifacts and select for high quality variants. For GATK, variant QC comprised filtering by QUAL >  = 30, a minimum depth per variant of 30 and removal of variants with strand bias p < 0.05. An additional filter was applied to exclude variants with < 20% and/or < 4 variant reads, to mimic LifeScope stringency filters. Variants were annotated using ANNOVAR (version 2019Apr09).