title: GSM8686063: K562_SSRP1_dTAG_Micro-C_S4Rep2; Homo sapiens; OTHER
library_name: GSM8686063
library_construction_protocol: 30 M cells per replicate were divided into 6 reactions each and crosslinked in phosphate buffered saline (PBS, Thermo Fisher Scientific) supplemented with 3 nM disuccinimidyl glutarate (DSG, Thermo Fisher Scientific) and 1% formaldehyde (Thermo Fisher Scientific). The reactions were quenched with 0.375 M Tris-HCl pH 7.5 (Thermo Fisher Scientific). For the following steps, all reactions were consecutively performed in the same tube. To prepare the samples for digestion, they were lysed in buffer cMB#1 (50 mM NaCl, 10 mM Tris, 5 mM MgCl2, 1 mM CaCl2, 0.2% NP-40, 1×Protease Inhibitor Cocktail (PIC)) at 4°C. Subsequent digestion with 34 Kunitz U of Micrococcal nuclease (MNase, NEB, M0247) per 1 M cells was performed for 20 min at 37°C. 4 mM ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA, Sigma-Aldrich, E3889) was added to quench the reaction. A 5% aliquot was assessed for digestion efficiency by de-crosslinking and extracting DNA using DNeasy blood and tissue kit (Qiagen). Samples with 80-90% mono-nucleosomal fragments were selected for further processing. For end-chewing, nuclei were subjected to 1×NEBuffer 2.1 (NEB), 2 mM adenosine 5'-triphosphate (ATP, Jena Bioscience), 5mM 1,4-dithiothreitol (DTT, Roche), and 0.1 U/μL T4 Polynucleotide Kinase (T4 PNK, NEB, M0201L) for 15 min at 37°C with mixing. 0.05 U/μL Klenow fragment (NEB, M0210L) was added and incubated for 15 min at 37°C with mixing. Fragment ends were labelled with biotin by adding 66 μM Biotin-14-dATP (Jena Bioscience), 66 μM Biotin-11-dCTP (Jena Bioscience), 66 μM ddGTP (Jena Bioscience), 66 μM ddTTP (Jena Bioscience), 1×T4 DNA ligase buffer (NEB), and 100 μg/mL bovine serum albumin (BSA, Sigma-Aldrich). The reaction was incubated for 45 min at 25°C with interval mixing and quenched with 30 mM ethylenediaminetetraacetic acid (EDTA, Sigma-Aldrich). For proximity ligation, biotinylated samples were incubated with 1×T4 DNA ligase buffer (NEB), 100 μg/mL BSA (Sigma-Aldrich), and 20 U/μL T4 DNA ligase (NEB, M0202L) for 2.5 h at 25°C. Subsequently, biotin-dNTPs from unligated ends were removed with 1×NEBuffer#1 (NEB) and 5 U/μL Exonuclease III (NEB, M0206L). The ligated product was de-crosslinked and purified using DNeasy blood and tissue kit (Qiagen, 69504). Di-nucleosomal fragments (~360 bp) were selected by a two-sided size selection using Mag-Bind TotalPure NGS beads (Omega Bio-tek). After pulldown of biotinylated fragments with Dynabeads MyOne Streptavidin T1 beads (NEB), the samples were indexed "on-bead" using NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB, E7645).