title: RC-seq: human brain
design: Libraries were processed prior to array hybridization by adding the following components to a 1.5ml tube: 320ng of library material, 0.64µl of 100µM Illumina primer PE 1.0 and PE 2.0 and 64µl of Roche NimbleGen proprietary capture enhancing (PCE) compound (in place of Cot-1 repetitive DNA blocker). Samples were dried down by puncturing a hole in the 1.5ml tube cap with a 20 gauge needle and processing in an Eppendorf Vacufuge (San Diego, CA) set to 60°C for 20 minutes. Samples were then re-suspended by adding 15.4µl of water and placing tubes on a heating block at 70°C for 10 minutes, followed by vigorous vortex mixing for 30 seconds and centrifugation to recollect any dispersed sample.   To each sample tube 25.6µl NimbleGen SC Hybridization Buffer (Part# 05340721001) and 10.24µl NimbleGen Hybridization component A (Part# 05340721001) were added, followed by vortexing for 30 seconds, centrifugation and placement on a heating block at 95°C for 10 minutes. Samples were again mixed for 10 seconds, spun down, and placed in a Roche NimbleGen Hybridization System at 42°C until ready for hybridization. The capture array was comprised of three identical sub arrays of 720K features targeting human retrotransposons (see above). To each slide, a NimbleGen HX3 mixer was affixed according to the manufacturer’s instructions and 16µl of the hybridization mixture (sample library, PCE, Ilumina primers, SC Hybridization Buffer and SC Component A) was pipetted into each of the three sub-array fields. Loading and vent holes were covered with port seals and each sample was hybridized for 72 hours at 42°C on Hybridization Station setting “B”. Slide washing and sample library elution were performed as previously described (Y. Fu et al., Plant J 62, 898).