title: GSM4555185: pCHi-C_iPax7+Dox_rep1; Mus musculus; Hi-C
library_construction_protocol: Approximately 30,000,000 iPax7 cells (+/-Dox) were cross-linked for 10 min by 2% formaldehyde in fresh media. Cross-linking was quenched by addition of 0.125M glycine. Cells were incubated at room temperature for 5 min and then centrifuged at 4°C for 5 min. Cross-linked cells were washed once in cold PBS, and the cell pellets were flash frozen in liquid nitrogen and stored at −80°C. Hi-C library generation was carried out with in-nucleus ligation as described previously (Nagano et al., 2015), with slight modifications. Briefly, chromatin was digested overnight at 37 °C with HindIII (NEB). Digested ends were filled in using biotinylated d-CTP and ligated in preserved nuclei. Cross-links were reversed by proteinase K treatment at 65 °C overnight and DNA was purified by phenol-chloroform extraction. DNA concentration was measured using Qubit 2.0 Fluorometer (Life Technologies). Biotin was removed from non-ligated restriction fragment ends by incubating the Hi-C library DNA with T4 DNA polymerase (NEB) for 4h at 20 °C in the presence of dATP and dGTP. After DNA purification with phenol:chloroform, 40 μg of DNA was sheared to an average size of 350 bp using Covaris LE220 following the manufacturer's instructions. Double size selection of DNA was performed using AMPure XP beads (Beckman Coulter) and the sonicated DNA was end-repaired with T4 DNA polymerase, T4 DNA polynucleotide kinase, Klenow (all NEB) and dNTPs in 1× T4 DNA ligase reaction buffer (NEB). Biotin-marked ligation products were isolated using MyOne Streptavidin C1 DynaBeads (Invitrogen), adenine-tailed and ligated to paired-end adaptors (Illumina). The immobilized Hi-C products were amplified using SureSelect Primers from SureSelectXT Reagent kit, HSQ (Agilent Technologies) for pCHi-C-seq with 7 PCR amplification cycles with Phusion High-Fidelity DNA Polymerase (NEB). Capture Hi-C of promoters was carried out with SureSelect target enrichment (SureSelectXT Custom 3-5.9Mb library, Agilent Technologies), using the custom-designed biotinylated RNA bait library that overlap 25,747 Ensembl-annotated promoters of protein-coding, noncoding, antisense, snRNA, miRNA and snoRNA transcripts (Schoenfelder et al., 2015) and custom paired-end blockers according to the manufacturer's instructions (Agilent Technologies). 500 ng to 1 μg of Hi-C library were used for capture. The enriched library was further amplified for pCHi-C-seq with 6 PCR amplification cycles using SureSelect primers (SureSelectXT Reagent kit, HSQ, Agilent Technologies) and Phusion High-Fidelity DNA Polymerase (NEB). Paired-end reads for pCHi-C libraries were obtained with Illumina NextSeq 500 (75 cycles).
attributes.tag: GEO Accession
attributes.value: GSM4555185