Bcl2fastq v2.17.1.14 conversion software

RNA was extracted from 0.1 g of frozen aerial parts of rice seedlings grown under 24-h Pi deficiency or control conditions using a RNeasy Plant Mini kit (Qiagen). The RNA-Seq libraries were prepared with a TruSeq Stranded mRNAseq Sample Prep kit (Illumina). The libraries were quantitated by qPCR and sequenced for 101 cycles from one end of the fragments on a HiSeq2500 using a HiSeq SBS sequencing kit version 4. Fastq files were generated and demultiplexed with the bcl2fastq v2.17.1.14 Conversion Software (Illumina). Illumina reads of all samples have been submitted to the Sequence Read Archive at the NCBI under accession number SRP102661. RNA-Seq reads for each sample were mapped to the reference genome (MSU Rice Genome Annotation Release 7.1) using the Bowtie2 tool (Langmead and Salzberg, 2012 (link)). To quantify transcript abundance, the Cuffdiff tool was applied to obtain fragments per kilobase of transcript per million mapped reads (FPKM) (Trapnell et al., 2012 (link)). Differentially expressed genes were identified using the DESeq2 package (FDR<0.001) (Love et al., 2014 (link)) and the normalized rLog (regularized Log-transformation) values of selected genes (n=2002) with FDR<0.001 obtained from DESeq2 were used for clustering. The fuzzy k-means clustering was done with the Aerie tool (Gasch and Eisen, 2002 (link)).

DNA amplicons for NGS were generated by PCR using KAPA HiFi HotStart (Roche), according to the manufacturer’s instructions, using primers with overhangs compatible with Nextera XT indexing (IDT, Supplementary Table S2). Following validation of the quality of PCR products by gel electrophoresis, the PCR products were isolated using AMPure XP PCR purification beads (Beckman Coulter). Indexed amplicons were then generated using a Nextera XT DNA Library Prep Kit (Illumina), quantitated, and pooled. Libraries were sequenced with a MiSeq Nano flow cell for 251 cycles from each end of the fragment using a MiSeq Reagent Kit v2 (500 cycles). FASTQ files were created and demultiplexed using bcl2fastq v2.17.1.14 Conversion Software (Illumina). Deep sequencing was performed by the Roy J. Carver Biotechnology Center at the University of Illinois at Urbana-Champaign.

Sequences were demultiplexed at the sequencing facility with the bcl2fastq v2.17.1.14 Conversion Software (Illumina, San Diego, CA), allowed 0 mismatches in the barcode sequences. De-multiplexed forward (read 1) and reverse reads (read 2) were further processed using the QIIME software package (27 (link)) The paired-end reads were merged, filtered and split into libraries as previously described (26 (link)). The representative operational taxonomic units (OTU) picking, chimera removing and construction of phylogenetic tree were performed as described by Monaco et al. (26 (link)). The representative sequence of each OTU was assigned to different taxonomic levels using Ribosomal Database Project naïve Bayesian rRNA Classifier (28 (link)) at 80% confidence level on the Greengenes reference database v.13.8. An OTU table was created and further filtered to remove non-aligned and chimeric OTUs and singletons. Alpha diversity (observed OTUs, Chao1 and Shannon and Simpson reciprocal indices) and beta diversity analysis was performed from the filtered OTU table after rarefying to 26,350 reads for each sample.