title: GSM8512762: ESCOBulk2, mESC, Rep2, bulk_Hi-C; Mus musculus; Hi-C
library_name: GSM8512762
library_construction_protocol: Bulk Hi-C library preparation in alignment with the preliminary processing steps of the single sperm cell Hi-C protocol, there are deviations in the subsequent procedures post-digestion. After inactivating MboI and cooling to room temperature. The sample was filled in the restriction fragment overhangs and mark the DNA ended with biotin, 20 μL of fill-in master mix (6 μL 1 mM Biotin-14-dATP (Jena Bioscience, NU-835-BIO14-S), 1.8 μL 10 mM dCTP/dGTP/dTTP mix, 4 μl 5 U μL−1 DNA polymerase I, large (Klenow) (NEB, M0210) and 8.2 μL water) was added. Samples were mixed by pipetting and incubated at 37 °C for 90 minutes. 72 μL ligation mix buffer (18 μL 10× T4 DNA ligase buffer, 1 uL 20 mg/mL BSA, 12 μL 1 U/uL T4 DNA ligase, and 41 μL water) was added and samples were incubated at 16 °C for 10 hours. After ligation, the tube was centrifuged at 2,500 g for 5 minutes at 4 °C, then the supernatant was removed and the pellet was resuspended in 200 μL reverse cross-link buffer (2 μL 1 M Tris pH 8.0, 4 μL 5 M NaCl, 2 μL 60 mg/mL Qiagen Protease, 0.4 μL 0.5M EDTA and 191.6 μL water) and incubated at 50 °C for 1 hour and 70 °C for 30 minutes. The sample was purified with DNA Clean & Concentrator-5 (ZYMO, D4014). To fragment the DNA ligation products, 50 ng of sample DNA was subjected to tagmentation using the TruePrep DNA Library Prep Kit V2 for Illumina (Vazyme, TD501), adhering to the manufacturer's protocol. Notably, only half of the enzyme quantity was utilized in the process and amplified only for 2 cycles. The sample underwent purification and subsequent elution with 20 μL of nuclease-free water. To ascertain the quantity of MyOne Streptavidin beads required for the library pull-down, a ratio of 2 μL MyOne Streptavidin C1 beads per 1 μg of Hi-C DNA was employed, with a minimum threshold set at 10 μL of MyOne Streptavidin C1 beads (Invitrogen, 65001). Here, we took 10 μL of magnetic beads for biotin pull-down. The washing of magnetic beads was performed according to the manufacturer's instructions. After the washing step, the beads were resuspended in 20 μL 2× Binding and Washing (B&W) buffer and mixed with 20 μL ligation products. The tube was incubated at room temperature for 15 minutes, then placed on a magnet for 3 minutes, and discarded the supernatant. Following two rounds of washing with 45 μL of 1× B&W buffer, the beads were subsequently resuspended in a 50 μL PCR reaction mixture (10 μL 5× KAPA HiFi GC Buffer, 1.5 μL dNTP Mix (10 mM each), 1 μL KAPA HiFi DNA Polymerase (1 U/μL), 5 μL N5XX primer, 5 μL N7XX primer (Vazyme, TD202) and 27.5 μL water) and subjected to amplification through a 10-cycle PCR protocol. The barcoded Hi-C library was then pooled and purified with 0.6× and 0.15× VAHTS DNA Clean Beads.