RNA-Seq of Homo sapiens: adult male esophagus
Illumina TruSeq Stranded Total RNA Kit with Ribo-Zero Gold Total RNA samples (100-500 ng) were hybridized with Ribo-Zero Gold to substantially deplete cytoplasmic and mitochondrial rRNA from the samples.  Stranded RNA sequencing libraries were prepared as described using the Illumina TruSeq Stranded Total RNA Kit with Ribo-Zero Gold (RS-122-2301 and RS-122-2302).  Purified libraries were qualified on an Agilent Technologies 2200 TapeStation using a D1000 ScreenTape assay (cat# 5067-5582 and 5067-5583).  The molarity of adapter-modified molecules was defined by quantitative PCR using the Kapa Biosystems Kapa Library Quant Kit (cat#KK4824).  Individual libraries were normalized to 10 nM and equal volumes were pooled in preparation for Illumina sequence analysis. HiSeq 50 Cycle Single-Read Sequencing version 4Sequencing libraries (25 pM) were chemically denatured and applied to an Illumina HiSeq v4 single read flow cell using an Illumina cBot.  Hybridized molecules were clonally amplified and annealed to sequencing primers with reagents from an Illumina HiSeq SR Cluster Kit v4-cBot (GD-401-4001).  Following transfer of the flowcell to an Illumina HiSeq 2500 instrument (HCSv2.2.38 and RTA v1.18.61), a 50 cycle single-read sequence run was performed using HiSeq SBS Kit v4 sequencing reagents (FC-401-4002).