title: RNA interactome:Halo capture
design: After proteinase K treatment, the RNA was dephosphorylated (Fast AP) andcyclic phosphates removed (T4 PNK) and then cleaned using Silane beads as previouslydescribed86. An RNA adapter containing a RT primer binding site was ligated to the 3 end of thecleaned and end-repaired RNA. The ligated RNA was reverse transcribed (RT) into cDNA, theRNA was degraded using NaOH, and a second adapter was ligated to the single stranded cDNA.Library preparation was the same for input samples except that an initial chemical fragmentationstep (90?C for 2 min 30 s in 1X FastAP buffer) was included prior to FastAP treatment. Thischemical fragmentation step was designed to be similar to the fragmentation conditions used forpurified Halo bound samples. The DNA was amplified and Illumina sequencing adaptors wereadded by PCR using primers that are complementary to the 3 and 5 adapters. The molarity ofPCR amplified libraries were measured by Agilent Tapestation High Sensitivity DNA screentapesand all samples were pooled at equal molarity. The pool was then purified and size selected on a2% agarose gel and cut between 150-700 nts