title: GSM2461047: HiC_NIV_ko_1; Mus musculus; OTHER
library_construction_protocol: For Hi-C, five thousands to millions cells were stained with Biotin anti-CD45.2 antibody. Cells were bound to biotin binder (Dynabeads Biotin Binder, Cat#11047, Invitrogen) and cross-link with 1% formaldehyde.  Biotin binders were blocked by 0.5mM biotin solution. Cells were lysed and digested with CviQ I + CviA II + Bfa I for 20min. For DNase-Seq, 1000 purified cells were lysed in 32ul of lysis buffer (10mM Tris-Cl, pH7.5, 10mM NaCl, 3mM MgCl2) and digested with 8ul of DNase I for 5 min at 37°C. The reaction was stopped by adding 40ul of stop buffer containing 10mM Tris-Cl, pH7.5, 10mM NaCl, 10mM EDTA, 2% SDS, 0.5mg/ml Proteinase K, and 1ng/ul of circular carrier DNA), followed by incubation at 65°C for 1 hour. For MNase-Seq, 100 purified cells were lysed in 32ul of lysis buffer (10mM Tris-Cl, pH7.5, 10mM NaCl, 2mM CaCl2) and digested with 8ul of MNase I for 5 min at 37°C. The reaction was stopped by adding 40ul of stop buffer containing 10mM Tris-Cl, pH7.5, 10mM NaCl, 10mM EGTA, 2% SDS, 0.5mg/ml Proteinase K, and 1ng/ul of circular carrier DNA), followed by incubation at 65°C for 1 hour. For RNA-Seq, 2ng of total RNA were extracted miRNeasy Micro Kit (50) from Qiagen (Cat # 217084). The Hi-C samples were processed following the Hi-C protocol (PMID 19815776) with modifications briefly described as follows: DNA end were marked by biotin-14-dATP with Klenow(larg) for 1hrs at 37°C. Blunt-end DNA were ligated with T4 DNA Ligase for overnight at 16°C. DNA were reverse cross-linked and purified by phenol/chloroform extraction. Biotin were removed from unligated DNA-ends by T4 DNA polymerase for 2hrs at 12°C. DNA were purified by phenol/chloroform. DNA were sheared to 300-500bps by sonication and flowed by DNA-end repair and add”A”34. Biotin labeled DNA were pull-downed by streptavidin beads and then flowed by illumine adapter ligation and PCR amplification. Library construction protocals for DNase-Seq, MNase-Seq and RNA-Seq follow that described in (PMID 26605532), (PMID 18329373) and (PMID 24056875), respectively.
attributes.tag: GEO Accession
attributes.value: GSM2461047