title: GSM8487842: Evf1_2 WT rep2 HiC; Mus musculus; Hi-C
library_name: GSM8487842
library_construction_protocol: HiCseq: HiC was performed using the Arima-HiC kit (A510008), according to the manufacturers protocols with modifications as described previously (Lafontaine et al., 2021, Curr Protoc. 2021;1(7):e198. doi:10.1002/cpz1.198). 12-16 E13.5 ganglionic eminences were isolated from 6-8 mouse embryonic brains and dissociated in L15 media gently first with a 1000 µL tip and then with a 200 µL tip 10-15 times to create a single cell suspension. Cells were spun for 2.5 minutes at 1000 rpm and 4°C. The L15 was aspirated and cells were resuspended in 500 µL of ice-cold Nuclear Extraction Buffer (0.32 M sucrose, 5 mM CaCl2, 3 mM Mg(Ac)2, 0.1 mM EDTA, 20 mM Tris-HCl pH 8.0, 0.1% TritonX-100) and incubated on ice for 10 minutes. Nuclei were pelleted at 100 for 2.5 minutes at 4°C. Nuclei Extraction Buffer was removed and nuclei were resuspended in 1X PBS. Nuclei were fixed in 10 mL of PBS with 1% formaldehyde with rotation for 10 minutes at room temperature. Crosslinking was then quenched with glycine to a final concentration of 125 mM for 5 minutes at room temperature. Nuclei were washed with 1x PBS and pelleted for 15 minutes at 500g and 4°C. Nuclei were then crosslinked with fresh 3 mM DSG (disuccinimidyl glutarate) in PBS for 40 minutes with rotation at room temperature. Crosslinking was quenched with 0.4 M glycine for 5 minutes, and then washed with 1X PBS. The nuclei were flash frozen in liquid nitrogen. The input amount was estimated following the Arima-HiC instruction protocol.  Nuclei were processed according to the Arima-HiC protocol, briefly: DNA was extracted using AMPure XP Beads and then quantified with Qubit. 75 ng of the sample was transferred to a new tube and used for quality control to determine the percentage of biotin labeling following the Arima-HiC protocol. Library preparation was performed using the Arima Library Prep for Arima HiC+ Kit (A510008, A303011) according to the manufacturer protocols. DNA was fragmented with Covaris for an average fragment size of 400bp. Volumes for the protocol were adjusted for an initial volume of 130 µL for DNA fragmentation. DNA was size selected with AMPure XP Beads and then enriched with biotin using the kit provided T1 beads. Library preparation was performed according to the protocol and a unique index pair was added to each sample before the samples were amplified with PCR and purified with AMPure XP Beads.