title: GSM1682604: n6_6_4C; Mus musculus; OTHER
library_construction_protocol: C57BL/6 male Mice of six weeks old were entrained to 12 hr light and 12 hr dark for one week and then switched to constant dark. Three mice were sacrificed in the dark at ZT6 and ZT18, respectively. Mice liver cells were quickly dispersed and filtered through the 40 mm cell strainer to make a single-cell suspension. Approximately 50-million cells were ﬁxed in 1% formaldehyde for 10 min at room temperature before being quenched with 0.125 M glycine. Cells were then lysed in cold lysis buffer (10 mM Tris HCl, 10 mM NaCl, 0.2% NP-40, 1×protease inhibitor) for 15 min on ice. After being washed twice, nuclei were re-suspended in Buffer 2.1 (New England Biolabs), including 0.1% SDS, and were incubated for 10 min at 65°C. 1% (final concentration) Triton X-100 was added to quench SDS and centrifuged to remove SDS and Triton. Nuclei were then digested overnight by 800U Hind III (NEB) at 37℃ with shaking. After inactivation by 1.6% (ﬁnal concentration) SDS at 65℃ for 20 min, samples were washed and re-suspended in ligation buffer, and ligated by 100U T4 DNA ligase (Thermo Fisher Scientific) at 16℃ for 4 h and then room temperature for 30 min. Ligated chromatin was digested by proteinase K before DNA purification. The purified DNA was further digested by Dpn II (NEB) and then circularized using T4 DNA ligase (Thermo Fisher Scientific). After puriﬁcation, 200ng of DNA from each sample was used as template for the PCR amplification using the DyNAzyme EXT (Finnzymes). Primers specific to bait were applied to amplify the interactome of interest in a 25ul reaction volume under the following PCR conditions: 1 cycle at 94°C for 2 min; (94°C 30 sec; 60°C 30 min; 72°C 2 min) ×18 cycles; 1 cycle of 72°C 7 min. 1uL of PCR products was used as template for a second PCR reaction utilizing the primers with the addition of the Illumina adaptors in a 50ul volume under the same PCR conditions. The PCR-amplified library was purified and sequenced with a 100bp read length using the Illumina HiSeq2000.
attributes.tag: GEO Accession
attributes.value: GSM1682604