title: GSM9004221: MicroC, Day7 Neuron, Aux Day0-7, rep2; Mus musculus; Hi-C
library_construction_protocol: NPCs and Day 7 In-vitro differentiated neurons were harvested, counted and collected in 10%FCS/1XPBS and double crosslinked using formaldehyde (ThermoFisher #28908) and DSG (disuccinimidyl glutarate, 7.7Å) (TCI, D5445), respectively. Following quenching of the fixation reactions and washes in ice-cold 1xPBS, 5M cell aliquots were pelleted and snap-frozen in liquid nitrogen for storage at −80°C. Cell membranes were solubilized to extract intact nuclei by resuspending crosslinked 5M cell pellets in Micro-C Buffer #1 (MB#1; 50 mM NaCl, 10 mM Tris-HCl pH = 7.5, 5 mM MgCl2, 1M CaCl2, 0.2% NP-40, 1x Protease Inhibitor Cocktail) at 1M cells per 100 μL for 20 min on ice. Following an MB#1 wash, samples were resuspended in 250 μL MB#1 and 1 μL of 20 U/μL MNase (Worthington Biochem #LS004798). The digestion reaction was thoroughly mixed and incubated at 37°C for 15 min on a thermomixer shaking 850rpm. MNase was inactivated by the addition of 200mM EGTA (4 mM final conc.) and incubating for 10 min at 65˚C. at this stage, a digestion efficiency determination step was performed: aliquots of digested and undigested samples were treated with Proteinase K for 1-2 hours at 65˚C, to reverse crosslink. After purification of the the DNA using ZymoClean DNA purification kit, the fragments size was measured on TapeStation, to ensure primarily (~80%) mononucleosomal fragments (150-200 bp). Digested nuclei were washed twice with ice-cold Micro-C Buffer #2 (50 mM NaCl, 10 mM Tris-HCl pH = 7.5, 10 mM MgCl2). To generate blunt ends on digested DNA fragments prior to proximity ligation and add biotinylated nucleotides, a series of enzymatic processing steps were performed. First, for addition of 5'-phosphate groups and removal of 3'-phosphate groups, digested samples were incubated with: 10xNEBuffer 2.1 (NEB, #B7202S), 100mM ATP (NEB, #P0756), 100mM DTT and 10U/μL T4 PNK (NEB, #M0201), at 37°C for 15 min with interval mixing. Then 50 U of DNA Polymerase I Klenow Fragment (NEB #M0210) was added to the reaction and incubated at 37°C for 15 min with interval mixing. Next, the following mixture was added to each sample: 10 mM dGTP and dTTP (NEB, #N0446), 1mM Biotin-dATP and Biotin-dCTP (Jena Bioscience, #NU-835-BIO14, #NU-809-BIOX), T4 DNA Ligase Buffer and BSA. The reactin was incubated for 45 min at 25°C with interval mixing and then quenched by 30 mM EDTA heat inactivate for 20 min at 65°C. Finally, the samples were washed once with cold Micro-C Buffer #3 (50 mM Tris-HCl pH 7.5, 10 mM MgCl2). Proximity ligation and Removal of unligated ends was performed by first washing the samples with 1x Ligase buffer, followed by resuspending in ligation reaction mix: 10x NEB Ligase buffer, 200xBSA, and 400U/ μl NEB T4 Ligase (NEB, #M0202L) and slowly rotating at RT for 2-3 h. Then, the chromatin was collected and removal of biotinylated dNTPs from all unligated fragment ends was performed by incubating the samples in 10x NEBuffer#1 (NEB # B7001) and 100U/ μl Exonuclease III (NEB # M0206), for 15 min at 37°C with interval mixing. Reverse crosslinking of the samples was performed by incubating with 20mg/ml Proteinase K, 10% SDS and 100mg/ml RNase A overnight at 65°C. After reverse crosslinking, Dinucleosomal DNA was purified in two steps: first, phenol:chloroform:isoamylic alcohol DNA extraction was performed. Then, the samples were loaded on 2% agarose gel. Band between 250 – 400 bp was cut out of the gel and then purified Zymo Gel Purification kit (Zymo Research #D4008). DNA amounts were Measured using Qubit™ High Sensitivity dsDNA kit. The biotin-bound ligated DNA contact fragments were pulled down using Dynabeads MyOne Streptavidin C1 (Invitrogen #65001) in TBW (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 2 M NaCl), and then washed once with 10mM Tris-HCl pH 7.5. For each micro-C sample, libraries were generated using the NEBNext Ultra II DNA Library Prep Kit (NEB, E7645), without size selection and with Pre-PCR wash of the beads with 100 μl 0.1xTE. PCR amplification of libraries was performed with 8 cycles of enrichment. Finally, libraries were purified using 0.9X AmPure XP beads (Beckman Coulter #A63880). Final libraries were quality controlled for size and concentration and sequenced on a NovaSeq Illumina sequencer with 100-bp paired-end reads.