title: RNA-Seq of monocytes after alcohol use
design: Total RNA was isolated from PBMC or CD14+ cells using the mRNeasy kit (Qiagen, Valencia CA) following manufacturer instructions and quality assessed using Agilent 2100 Bioanalyzer. Libraries from PBMC RNA were generated using the TrueSeq Stranded RNA LT kit (Illumina, San Diego, CA, USA). Libraries from CD14+ RNA were generated using the NEBnext Ultra II Directional RNA Library Prep Kit for Illumina (NEB, Ipswitch, MA, USA), which allows for lower input concentrations of RNA (10ng). For both library prep kits, rRNA depleted RNA was fragmented, converted to double-stranded cDNA and ligated to adapters. The roughly 300bp-long fragments were then amplified by PCR and selected by size exclusion.  Libraries were multiplexed and following quality control for size, quality, and concentrations, were sequenced to an average depth of 20 million 100bp reads on the HiSeq 4000 platform.