repair kit (Epicenter Cat. No. ER0720), and single 3′-adenine overhangs were added using Klenow(3′-5′ exo-) (New England Biolabs, Cat. No. M0212L). Qiagen MiniElute spin columns were used to clean up each of these reactions. Barcoded Illumina adapters prepared by the Broad Institute’s Genomic’s platform, were ligated to cleaned DNA fragments with DNA ligase (New England Biolabs, Cat. No. M2200S) and subsequently cleaned using 0.7X AMPure XP beads with 70% freshly prepared ethanol washes two times. The libraries were then amplified by PCR using PFU ULTRA II HS 2X Master Mix (Agilent Cat. No. 600852). Size selection was carried out by cutting the section between 275 bp-600 bp after running electrophoresis on a 2% agarose gel using a 100 bp ladder (NEB-N3231S). The final library was extracted from the excised gel fragments using the Gel Extraction Kit (Qiagen-28706). Libraries were quantified by Qubit in triplicate and pooled for sequencing in 4-plex or 8-plex and sequenced for 36 bp single end reads on Illumina’s HiSeq2000 splitting the two cohorts across the pools as evenly as possible and targeting about 20M reads per sample.
