High throughput hiseq 2500

RNA was extracted using Trizol (Invitrogen). RNA samples were sent to the Genomics Research Center of the University of Rochester for RNA-Seq. The TruSeq RNA Sample Preparation Kit V2 (Illumina) was used for library construction. The libraries were hybridized to the Illumina single end flow cell and amplified using the cBot (Illumina) at a concentration of 8 pM per lane. Single end reads of 100 nt were generated for each sample. The sequencing was performed using the Illumina high-throughput HiSeqTM 2500. Normalization and differential expression analysis were carried out using DESeq2^{46 (link)}. Ontology and pathway analyses were performed using Enrichr program^{26 (link)}. TFBS enrichment analysis was done using oPOSSUM program^{27 (link)}. The parameters used in the oPOSSUM were as follows: conservation cut-off = 0.4, matrix score threshold = 85%, analyzed sequences = 2 kb upstream of transcription start sites, Z-score cut-off = 10, Fisher score cut-off = 7.

RNA was extracted using an RNeasy Mini kit (Qiagen, Valencia, CA) per manufacturer's instructions. High-quality RNA samples (pre-assessed by Nanodrop 2000) were further processed in the Genomics Research Center of the University of Rochester. Briefly, RNA quality assessed with the Agilent Bioanalyzer (Agilent, Santa Clara, CA). The TruSeq RNA Sample Preparation Kit V2 (Illumina, San Diego, CA) was used for next generation sequencing library construction per manufacturer's protocols. mRNA was purified from 100 ng total RNA with oligo-dT magnetic beads and fragmented. First-strand cDNA synthesis was performed with random hexamer priming followed by second-strand cDNA synthesis. End repair and 3′ adenylation was then performed on the double stranded cDNA. Illumina adaptors were ligated to both ends of the cDNA, purified by gel electrophoresis and amplified with PCR primers specific to the adaptor sequences to generate amplicons of approximately 200-500 bp in size. The amplified libraries were hybridized to the Illumina single end flow cell and amplified using the cBot (Illumina, San Diego, CA) at a concentration of 8 pM per lane. Single end reads of 100 nt are generated for each sample and aligned to the organism specific reference genome. The sequencing was performed using the Illumina high-throughput HiSeqTM 2500.