title: GSM1650022: DRIP_RNAseq_Control; Mus musculus; OTHER
library_construction_protocol: Genomic DNA was extracted from mouse ESC and was fragmented using HindIII, EcoRI, BsrGI, XbaI and SspI. Monoclonal S9.6 antibody was used to enrich for RNA/DNA hybrids. The immunoprecipitated material (with and without RNase H treatment) were denatured at 94°C for 1 min and cooled on ice. To reduce DNA background, the samples were treated with DNaseI at 37°C for 30 minutes and RNA was purified using Phenol/Chloroform/Isoamylalcohol extraction. 38 pmol of adaptor 1 (CTGAACCGCTCTTCCGATCTNNNNNN) was combined with 50 ng of S9.6 enriched RNA for first-strand cDNA synthesis with a Superscript III Reverse Transcription Kit (Life Technologies). After first-strand cDNA synthesis, RNA was degraded by sodium hydroxide and cDNA was purified by SILAN beads (Life Technologies). To preserve strand information, Adaptor 2 (5’-p-NNACGTAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT-3’ddC) was ligated to the 3’ end of first-strand synthesized cDNA. The ligation material was purified by SILAN beads and then PCR amplified with Illumina primers using 18 cycles of PCR. To remove PCR primers, libraries were purified by AMPure XP beads (Beckman Coulter).