title: GSM6423084: Epithelium cell_rep2; Mus musculus; OTHER
library_construction_protocol: Two independent biological replicates (5-10 x106) were crosslinked with disuccinimidyl glutarate (DSG) and formaldehyde. DNA in isolated nuclei were digested with AluI. A biotinylated bivalent linker was ligated to chromatin-associated RNA (with the ssRNA stretch on the linker) and nearby fragmented genomic DNA and captured by streptavidin microbeads for library construction. To amplify each library, 20 μl of PCR amplification mix (9.4 μl of H2O, 5 μl of DNA sample, 4 μl of 5× Phusion HF Buffer, 40 pmol dNTP, 5 pmol Primer#1, 5 pmol Primer#2, 0.4 U Phusion High-Fidelity DNA Polymerase (Thermo Fisher)) was prepared. PCR primers consist of Primer#1 (5′-AATGATACGGCGACCACCGAGATCTACACBBBBBACACTCTTTCCCTACACGACGCTCTTCCGATCT-3′ (BBBBB: 5 nt barcode for multiplexing libraries)) and Primer#2 (5′-CAAGCAGAAGACGGCATACGAGACGTGTGCTCTTCCGATCT-3′). PCR was performed initially for 30 seconds at 98°C, and then 16 cycles of 10 sec denaturation at 98°C, 30 sec annealing at 65°C, and 15 sec extension at 72°C. The PCR product was resolved by native 10% polyacrylamide gel and the band of 194 bp in size was recovered.Sequencing was performed on HiSeq400 (Illumina, 200 million reads/sample).