Truseq mrna library preparation kit

The METABRIC dataset of tumor samples included 2433 samples from the METABRIC project^{14 (link)} with DNA sequencing data, among which 480 were subjected to a capture-based RNA sequencing study^{23 (link)}. Sequencing libraries were generated as previously described. In brief, sequencing libraries using total RNA generated from frozen tissues with a TruSeq mRNA Library Preparation Kit using poly-A-enriched RNA (Illumina, San Diego, CA, USA) and enriched with the human kinome DNA capture baits (Agilent Technologies, Santa Clara, CA, USA). Six libraries were pooled for each capture reaction, with 100 ng of each library, and sequenced (paired-end 51bp) on an Illumina HiSeq2000 platform. We selected a subset of samples with DNA and RNA sequencing data and PIK3CA missense mutations for further analysis.
The TCGA dataset comprised 695 samples from TCGA breast cancers^{35 (link)}, from which we selected a subset of 289 samples with PIK3CA missense mutations for further analysis. Supplementary Table 3 summarizes the demographic features and disease characteristics of the two datasets.

Agrobacterium‐infiltrated N. benthamiana leaves were harvested after 24 h (T1) or 72 h (T2) and frozen in liquid nitrogen. Total RNA was extracted as described above and sequencing libraries were prepared using the TruSeq mRNA library preparation kit (Illumina) and sequenced on a HiSeq2000 (Illumina) using 2 × 100 bp paired‐end sequencing. Each treatment had three biological replicates, with each replicate being a pool of leaves from individual plants. The libraries were multiplexed and run on three lanes generating 48–54 million reads per sample. Resulting reads were then quality trimmed where fastq‐mcf (ea‐utils.1.1.2‐806) was used for adapter removal with a quality threshold cut‐off of 20, followed by the use of an in‐house perl script to trim 15 bases of the 5′‐end and remove any reads with N's or mononucleotides. Thereafter, reads were aligned to the Solgenomics N. benthamiana genome v.1.0.1 (https://solgenomics.net/organism/Nicotiana_benthamiana/genome) using Bowtie2 (v.2.2.5). The number of reads aligning to the annotated genes was counted using HTSeq (v.0.6.1p1). Differentially expressed genes were identified using DESeq2 analysis with a cut‐off probability of P < 0.05. Gene ontology (GO) enrichment analysis was performed using david (Huang et al., 2009) with TAIR10 annotation. The GOplot R package was used to visualise the GO enrichment analysis (Walter et al., 2015).

Total RNA was extracted with the RNAqeuous 4PCR Kit (Ambion) according to manufacturer’s instructions, with the addition of an initial 1-minute bead beating step using acid-washed sterile 425- to 600-μm glass beads (Sigma Aldrich) to ensure cells were mechanically removed and disrupted from the filter. Samples were eluted in 40 µL of sterile H₂O and stored in −80°C. Residual genomic DNA was removed by incubating RNA with deoxyribonuclease (DNase) I at 37°C for 45 minutes and purified by DNase I inactivation reagent (Life Technologies). Some samples required multiple incubations with DNase I. Sample concentrations and RNA integrity numbers (RINs) were determined using an Agilent Bioanalyzer 2100. RIN values were between 3.9 and 7.4. mRNA libraries were generated with ca. 2 µg of total RNA, using a poly-A selection primarily selecting mRNA of the eukaryotic plankton community, and prepared with the Illumina TruSeq mRNA Library Preparation Kit. Samples were individually barcoded and pooled prior to sequencing on a single lane of the Illumina HiSeq 4000 platform at Genewiz Sequencing Facility (S. Plainfield, NJ). Sequencing resulted in ca. 15 million 2 × 150 bp paired-end reads per sample (Table S3). Sequences were submitted to the NCBI Sequence Read Archive under accession number PRJNA877830.