Truseq sbs v3 reagents

WGS on genomic DNA samples was performed by Illumina, Inc. (San Diego, CA, USA) using TruSeq SBS v3 Reagents, HiSeq Control Software (HCS) and RTA on a HiSeq 2000 machine for real‐time image analysis and base calling. Genome assembly, genotype calling, and QC filtering was performed using tools in the CASAVA package. Multisample VCF files were generated using VCFtools (Danecek et al. 2011) and were backfilled with custom scripts to include homozygous reference genotypes and depth of coverage. Full details of the sequencing, alignment, and variant calling process are provided in the supporting information of Mathias et al. (2016).

RNA sequencing analysis was performed in 24 samples. The SMARTer Ultra Low Input RNA Kit for Sequencing v4 (Clontech Laboratories) was used to generate first-strand cDNA from 750 pg total-RNA. Double-stranded cDNA was amplified by LD PCR (12 cycles) and purified via magnetic bead clean-up. Library preparation was carried out as described in the Illumina Nextera XT Sample Preparation Guide (Illumina). 150 pg of input cDNA was tagmented (tagged and fragmented) by the Nextera XT transposome. The products were purified and amplified via a limited-cycle PCR program to generate multiplexed sequencing libraries. For the PCR step 1:5 dilutions of index 1 (i7) and index 2 (i5) primers were used. The libraries were quantified using the KAPA SYBR FAST ABI Prism Library Quantification Kit (Kapa Biosystems). Equimolar amounts of each library were pooled, and the pools then used for cluster generation on the cBot with the Illumina TruSeq SR Cluster Kit v3. The sequencing run was performed on a HiSeq 1000 instrument using the indexed, 50 cycles single-read (SR) protocol and TruSeq SBS v3 Reagents according to the Illumina HiSeq 1000 System User Guide. Image analysis and base calling resulted in bcl files, which were converted into fastq files with the bcl2fastq v2.18 software. The sequencing data are available in the Gene Expression Omnibus database under accession number GSE179568.

Total RNA was isolated using TRIzol reagent (Biotopped, Beijing, China). Polyadenylated mRNA was purified using the Dynabeads® mRNA Purification kit (Invitrogen, Carlsbad, CA) and library construction was performed with the TruSeq RNA Sample Preparation Kit (Illumina, San Diego, CA) following manufacturer's instructions. Sequencing was conducted on the Illumina HiSequation 2500 system with TruSeq SBS v3 reagents (38 (link)). For each sample, the resulting sequences were trimmed based on quality scores and mapped to the B73 maize genome (ZmB73_RefGen_v4) by using Hisat2 (version 2.0.5) (39 (link)) with parameters: -dta, -score-min L,-0.6,-0.6. Only uniquely mapped reads were taken for the subsequent analysis.

Tissues preserved in RNAlater were homogenized in TRIzol (ThermoFisher Scientific) using a gentleMACS dissociator (Miltenyi Biotec Inc). Total RNA was isolated using the miRNeasy Mini kit (Qiagen) according to manufacturer’s protocols, including the optional DNase digest step. RNA quality and concentration were assessed using the RNA 6000 Nano LabChip assay on the 2100 Bioanalyzer instrument and Nanodrop 2000 spectrophotometer (Thermo Scientific). Prior to 2016, non-stranded libraries were constructed using TruSeq RNA Library Prep Kit v2 (Illumina). Stranded libraries were prepared using the KAPA mRNA HyperPrep Kit (KAPA Biosystems), according to the manufacturer’s instructions. Briefly, the protocol entails isolation of polyA containing mRNA using oligo-dT magnetic beads, RNA fragmentation, first and second strand cDNA synthesis, ligation of Illumina-specific adapters containing a unique barcode sequence for each library, and PCR amplification. Libraries were checked for quality and concentration using the DNA 1000 assay (Agilent Technologies) and quantitative PCR (KAPA Biosystems), according to the manufacturers’ instructions. Libraries were pooled and sequenced 75 bp paired-end on the NextSeq 500 (Illumina) using NextSeq High Output Kit v2 reagents (Illumina), or 100 bp paired-end on the HiSeq2500 (Illumina) using TruSeq SBS v3 reagents (Illumina).

DNA fragments were end repaired, 3′‐adenylated and ligated to indexed adapters without size selection using Nextflex reagents (Newmarket Scientific, UK). Libraries were amplified with eight cycles PCR using Kapa HiFi PCR master mix (Anachem), primers removed with GeneRead size selection protocol (QIAgen) before quantification by Bioanalyser DNA 7500 assay. Libraries were pooled, denatured and diluted to 6 nM before clustering in a single lane of a high‐output Illumina flow cell. Sequencing (100 nt) was undertaken on a HiSeq 2500 using TruSeq SBS v3 reagents (Illumina).

Following library quantitation, DNA libraries were denatured, diluted, and clustered onto v3 flow cells using the Illumina cBot™ system. cBot runs were performed based on the cBot User Guide, using the reagents provided in Illumina TruSeq Cluster Kit v3. Clustered v3 flow cells were loaded onto HiSeq 2000 instruments and sequenced on 100 bp paired-end, non-indexed runs. All samples were sequenced on independent lanes. Sequencing runs were performed based on the HiSeq 2000 User Guide, using Illumina TruSeq SBS v3 Reagents. Illumina HiSeq Control Software and Real-time Analysis were used on HiSeq 2000 sequencing runs for real-time image analysis and base calling.