Unique dual indexes

ATAC-seq libraries were produced by the Applied Genomics, Computation and Translational Core Facility at Cedars Sinai. Briefly 50,000 BMDMs per sample were lysed to collect nuclei and treated with Tn5 transposase (Illumina) for 30 min at 37°C with gentle agitation. The DNA was isolated with DNA Clean & Concentrator Kit (Zymo) and PCR amplified and barcoded with NEBNext High-Fidelity PCR Mix (New England Biolabs) and unique dual indexes (Illumina). The ATAC-seq library amplification was confirmed by real-time PCR, and additional barcoding PCR cycles were added as necessary while avoiding overamplification. Amplified ATAC-seq libraries were purified with DNA Clean & Concentrator Kit (Zymo). The purified libraries were quantified with Kapa Library Quant Kit (KAPA Biosystems) and quality assessed on a 4200 TapeStation System (Agilent). The libraries were pooled based on molar concentrations and sequenced on a HiSeq 4000 platform (paired end, 100 bp).

The 12 mono- and dikaryotic strains (Fig. 2 and SI Appendix, Table S1) were grown in 2% liquid malt extract for 10 to 14 d on a rotary shaker. For Illumina sequencing, DNA was obtained from resulting mycelia and their source fruiting bodies using the Zymo Fungal/Bacterial miniprep kit. Sequencing libraries were prepared from 1 μg DNA using the TruSeq PCRfree DNA sample preparation kit (cat#20015962/3, Illumina) and unique dual indexes (cat#20022370, Illumina), targeting an insert size of 350 bp. The library preparation was performed according to the manufacturer’s instructions (guide #1000000039279). Sequencing was done using a NovaSeq 6000 SP flowcell, with paired-end 150-bp read length and v1 sequencing chemistry.
For Nanopore, we extracted and sequenced DNA only from the cultured strains. We used the Qiagen Genomic Tip 100/G following the manufacturer’s instructions, and isolated DNA was size selected for 25 kb or longer fragments using the Circulomics Short Read Eliminator Kit. No shearing was performed. Sequencing was done using two flowcells of the Nanopore PromethION system, base calling with ont-guppy-for-minknow 3.2.10, and reads were separated based on barcode using NanoComp.

Library preparation and sequencing were performed at the SNP&SEQ Technology Platform (Uppsala, Sweden). Sequencing libraries were prepared from 500 ng total RNA using the TruSeq Stranded mRNA Library Preparation Kit (cat# 20020595, Illumina Inc., USA), including polyA selection, following the instructions of the manufacturer (protocol #1000000040498). Unique dual indexes (cat# 20022371, Illumina Inc., USA) were used. The libraries were sequenced using a NovaSeq 6000 system (Illumina Inc., USA) and a SP-200 flow cell with pair-end 100 bp read length and v1.5 sequencing chemistry. A sequencing library for the phage PhiX was included as a 1% spike-in in the sequencing run. The sequencing generated a coverage of 8 to 14 M reads per library.