GSM8340296: BMDM_peak68866_0h; Mus musculus; OTHER
We crosslinked the lipid-A treated BMDMs with 1% formaldehyde for 10 min and quenched them with 2M Tris (0.125M final concentration) for 10 min at room temperature. Cells were lysed for 30 min in 1mL cold lysis buffer (10mM Tris-HCl pH 8.0, 10mM NaCl, and 0.2% NP-40 supplemented with protease inhibitor). Nuclei were pelleted and resuspended in 500μL of the first restriction enzyme buffer with 0.1% SDS and incubated for 1 hr at 37oC at 1200 rpm. Triton-X 100 (final concentration 0.8%) was then added, following incubation for 1 hr. 400 units of the first restriction enzyme (DpnII) was then added to the samples, incubated at 37oC for 6 hr, following by addition of another 400 units of enzyme for overnight digestion. The first restriction enzyme was inactivated by incubating at 65oC for 10 min and samples were placed on ice immediately. Ligation buffer (66mM Tris-HCl pH 7.5, 5mM MgCl2, 5mM DTT, 1mM ATP) was added to dilute samples to a total volume of 7mL. 2,000 units of T4 DNA ligase was added to each sample and incubated at 16oC overnight to achieve proximal ligation. Reverse crosslinking and RNA digestion were performed and followed by phenol/chloroform DNA extraction. Ligation efficiency was confirmed by running the samples before and after ligation on a 1% agarose gel. The ligated DNA was digested overnight in a volume of 500μL with 200 units of the second restriction enzyme (CviQI). The digested samples were diluted in the ligation buffer into a total volume of 7mL. Proximal ligation was performed by adding 4000 units of T4 DNA ligase, followed by overnight incubation at 16oC. The 4C template DNA was extracted by phenol/chloroform and ethanol precipitation. The extracted DNA was further purified using the QIAquick PCR Purificatioin kit. To generate the library for subsequent sequencing, reading and non-reading primers were designed for each viewpoint. Choice-Taq™ DNA Polymerase was used to PCR amplify 4C DNA templates containing the viewpoints in four of 25μL reactions. The PCR products were purified with the illustra™ GFX PCR DNA and Gel Band Purification Kit to remove primer dimers. Libraries were sequenced for 50-bp single-end reads on the Illumina Hiseq2000 platform.