Hiseq 2000 sequencer

RNA was isolated as described above. RNA-seq was performed by the Sequencing and Microarray Facility (SMF) core at MD Anderson. Libraries were generated using Illumina’s TruSeq kit and were sequenced using the Illumina HiSeq2000 Sequencer. Raw sequencing data (BCL format) were converted to Fastq files using Illumina Casava software (v1.8.2) and aligned to the mouse reference genome (mm10) using STAR software. The HTSeq-count program was used to generate raw read counts for each gene. The R package edgeR was used for data normalization and differential expression analysis using the criteria of log2 fold change ≥ 0.5 or ≤−0.5 and p value ≤ 0.1. Pathway enrichment analysis was performed using GSEA software based on p value from the aforementioned differential expression analysis, and clustering visualization was done by the R package heat map. To analyze YAP signature genes, the list of YAP signature genes 29 was obtained and formatted as the compatible pathway database (grp format) with GSEA. RNA from mouse colonic epithelial cells were isolated and sequenced using the Illumina HiSeq2000 Sequencer. In addition, we obtained a 92 IBD gene signature panel (common between the colons of ulcerative colitis and Crohn’s disease patients).

Deep sequencing of virus populations was performed using the Illumina HiSeq2000 sequencer with the sequencing kit, version 5. After removal of the four nucleotide bar codes used for multiplexing, more than 4 million single-end 76-nucleotide (nt) reads were obtained for each sample. Deep sequencing of selected virus mutants was performed with the Illumina HiSeq2000 sequencer using the HiSeq sequencing kit TruSeq v3. In these cases, more than 20 million paired-end 46-nt reads were obtained.

As an orthogonal validation, all samples were concurrently sequenced by conventional bulk next-generation sequencing (bulk-seq) using target-capture deep sequencing (N = 111, median coverage: 421×, IQR: 319×–610×) or whole-exome sequencing (N = 12, median coverage: 150×, IQR: 86×–160×). Target-capture next-generation sequencing was performed using a SureSelect (Agilent Technologies) custom panel of 297 genes that are recurrently mutated in hematological malignancies (Supplementary Table 3). Briefly, genomic DNA was extracted using an Autopure extractor (QIAGEN/Gentra) and was fragmented and bait-captured in solution according to the manufacturer’s protocols. Captured DNA libraries were then sequenced using a HiSeq 2000 sequencer (Illumina) with 76-bp paired-end reads. Whole-exome sequencing was performed using SureSelect V4 exome probes (Agilent Technologies) and a HiSeq 2000 sequencer (Illumina) with 76-bp paired-end reads. Modified Mutect and Pindel algorithms were used for mutation calling^{12 (link)}.

For WGS datasets, two separate library preparation protocols were used. The gDNA libraries for full genome libraries were prepared using the reagents from a TrueSeq DNA Sample Prep Kit according to the manufacturer’s instructions (TrueSeq DNA Sample Preparation Guide, revision C; Illumina Inc., San Diego, CA) with minor modifications. After the ligation, the first protocol uses a gel-free method for samples instead of a gel step that was used for the second protocol. Furthermore, the number of PCR cycles in the PCR enrichment step differs between the two protocols (five and ten cycles, respectively). A High Sensitivity DNA chip (Agilent Technologies 2100; Santa Clara, CA) was used for quantification and samples were subsequently sequenced on an Illumina HiSeq 2000 sequencer at the same laboratory.
Libraries for the WES samples were prepared using the Agilent SureSelect Kit (Agilent Technologies, Santa Clara, CA), Nimblegen Capture Kit V2 or Nimblegen Capture Kit V3 (Roche NimbleGen Inc., Madison, WI), according to the manufacturer’s instructions. A High Sensitivity DNA chip (Agilent Technologies 2100) was used for the quantification and the samples were subsequently sequenced on an Illumina HiSeq 2000 sequencer at the same laboratory.
The library preparation and sequencing of all RNA-Seq samples are described elsewhere [23 (link),24 (link)].

Total genomic DNA was extracted from leaf tissues using the DNeasy Plant Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions. Total RNA was extracted from whole plants using the RNeasy Plant Mini Kit (Qiagen). For the small noncoding RNA library, total RNA was extracted from leaves using the mirVana Kit (Ambion, Austin, TX, USA). The quality of the RNA and DNA was checked on a Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA). The libraries were prepared and sequenced according to the manufacturer's instructions (Illumina, San Diego, CA, USA). The DNA library was constructed using TruSeq DNA sample preparation kits and a single lane of an Illumina HiSeq2000 sequencer (PE, 2×101 bp). For the mRNA library, multiplex libraries were obtained using TruSeq RNA sample preparation kits, and the samples were sequenced in one lane of an Illumina HiSeq2000 sequencer (PE, 2×101 bp). The small RNA library was constructed using the TruSeq Small RNA Sample Prep Kit; the resulting single end library was sequenced in one lane of an Illumina GAIIX sequencer (SE, 1×35 bp). The files containing the sequences and quality scores of reads were deposited in the NCBI Short Read Archive, and the accession numbers are SRX465632 (genomic DNA-Seq), SRX465633 (mRNA-Seq), and SRX465634 (Small RNA-Seq).