Instrument: Illumina HiSeq 3000
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: PAIRED
Construction protocol: The epithelium cell pellet was lysed and total RNA was extracted by using RNeasy plus mini kit (QIAGEN, Germantown, MD) according to manufacturer's protocol. RNA concentration was determined on Qubit 2.0 Fluorometer (ThermoFisher, Waltham, MA) and RNA quality was assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). Total RNA with 28S/18S > 1 and RINs between 7.1 and 9.3 were used for RNAseq library construction. Kit for Illumina (New England Biolabs, Ipswich, MA) following manufacturer's instructions. Briefly, 500 ng of total RNA was used for mRNA isolation using NEBNext Poly(A) mRNA Magnetic Isolation module (New England Biolabs, Ipswich, MA). Purified mRNA was used to construct RNA library using NEBNext Ultra II Directional Library Prep kit (New England Biolabs, Ispwich, MA). Briefly, RNA was fragmented in NEBNext First Strand Synthesis Buffer by heating at 94 °C. This step was followed by first strand cDNA synthesis using reverse transcriptase and oligodT primers. Subsequently, synthesis of double-stranded cDNA was performed, followed by end-repair and adaptor ligation. At this point, Illumina adaptors were ligated to the sample. Finally, the library with unique barcode was enriched by 10 cycles of amplification, and purified by Agencourt AMPure beads (Beckman Coulter, Brea, CA). The library was sized on the bioanalyzer and quantitated by Qubit. In preparation for sequencing on the HiSeq3000 Illumina sequencer, uniquely barcoded libraries were normalized to 2.5 nM and equimolar amounts pooled. The RNA-seq library pool was spiked with 1% of PhiX library. Library pools were processed according to the Illumina HiSeq3000 protocol for clustering on the cBOT machine. After denaturation, neutralization and mixing with the ExAmp reagent, the final pool concentration for clustering was 0.25 nM. Sequencing was done using a 2x101 cycles format (paired-end configuration). A typical sequencing run in the HiSeq3000 produced 600-650 million paired-end reads per lane.