GSM7720756: BEAS2B_veh-2; Homo sapiens; OTHER
Micro-C was performed as described by Hsieh et al (PMCID: PMC7703524). The cell pellet was resuspended in Micro-C Buffer #1 (MB #1: 50 mM NaCl, 10 mM Tris-HCl-pH 7.5, 5 mM MgCl2, 1 mM CaCl2, 0.2% NP-40, 1X Protease Inhibitor Cocktail) and immediately divided equally into 2 replicate samples containing ~10 million cells each prior to incubation on ice for 20 min to release intact nuclei. Chromatin was fragmented using Micrococcal Nuclease (New England Biolabs) at a final concentration of 2000 gel units/ml (1:1000 dilution) in MB #1 and incubating at 37°C for 20 min with shaking at 850 rpm. Fragmented chromatin ends were repaired, blunted and biotinylated using biotin-ATP and -CTP (obtained from Axxora) and crosslinked nucleosomes were ligated by T4 DNA Ligase (New England Biolabs) for 2.5 hr at room temperature. Fragment ends were cleaned with Exonuclease III (New England Biolabs) and crosslinks were reversed by overnight incubation in 2 mg/ml Proteinase K (Sigma) in 1% SDS at 65C. Chromatin was then phenol:chloroform:isoamyl alcohol extracted, ethanol precipitated, treated with RNase A (Invitrogen), and purified with a DNA Clean and Concentrator-5 Kit (Zymo) prior to separation on a 3.5% NuSieve Agarose Gel (Lonza). The band containing the ligated di-nucleosomal DNA (~250-400 bp) was excised from the gel and DNA purified for library preparation using a ZymoGel Purification Kit (Zymo). Biotinylated di-nucleosomal DNA was captured using DYNAL MyOne Dynabeads Streptavidin C1 (Invitrogen) and used as input for the NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs), which includes standard procedures for end-repair, A-tailing, and adaptor ligation. Five cycles of initial library amplification employing KAPA HiFi HotStart ReadyMix (KAPA Biosystems) and Multiplex Oligos for Illumina, Index Primers Set 1 (New England Biolabs; for i7 barcoding) were followed by quantitative PCR assessment of 10% of the pre-amplified product to determine the lowest number of additional PCR cycles for sufficient final library amplification with minimal PCR duplication. Pre-amplified libraries underwent 8 additional cycles of amplification and were then size-selected and purified using Ampure XP Beads (Agencourt) at 0.9X volumes. Libraries were pooled and sequenced on an Illumina NovaSeq 6000 instrument using paired-end 2x150 bp reads by the Genomics Shared Resource at the University of Colorado Anschutz Medical Campus.