GSM6052323: LGE_rep1; Mus musculus; Hi-C
To recover embryonic tissue, dams were anesthetized with isoflurane and then euthanized by cervical dislocation. Embryos were removed from the uterus and kept in chilled artificial cerebrospinal fluid (ACSF, in mM: 87 NaCl, 26 NaHCO3, 2.5 KCl, 1.25 NaH2PO4, 0.5 CaCl2, 7 MgCl2, 10 glucose, 75 sucrose, saturated with 95% O2, 5% CO2, pH 7.4). Brains were extracted from E12.5 embryos, hemisected, microdissected to obtain the MGE, LGE, CGE and somatosensory cortex, and kept in ACSF. To collect whole cells, embryonic MGE, LGE, CGE and cortex tissue was incubated in 1 mg/mL Pronase (Roche #10165921001) in ACSF for 20 minutes at RT. Pronase solution was removed and 2 ml of reconstitution solution (1% fetal bovine serum + DNAse (1:10,000, Roche #47167280001)) in oxygenated ACSF was added. DNAse was not included in the reconsititution solution. Cells were triturated sequentially with fire-polished large, medium and small bore Pasteur pipettes to mechanically dissociate tissue. After cell dissociation, 1 million cells per region were fixed with 1% formaldehyde (Thermo: 28908) made in 1 ml HBSS media. Fixation was carried out at room temperature on a nutator, for 10 mins, protected from light. To stop fixation, glycine was added at a final concentration of 0.13 M and samples were incubated for 5 mins 595 at room temperature followed by 15 mins on ice. Fixed cells were then washed once with ice-cold PBS. After spinning cells at 2500g 4oC for 5 mins, the pellet was flash frozen in liquid nitrogen and stored at -80oC. To perform Hi-C, pellets were first thawed on ice and then incubated with 1 ml lysis buffer (10 mM Tris-HCL pH8, 10 mM NaCl, 0.2% Igepal CA-630, Roche Complete EDTA-free Sigma #11836170001). After lysis, cells were dounced and washed with cold PBS. Nuclei extracted with this method were then collected by centrifugation and subjected to DpnII digest in 50 μl 0.5% SDS and incubated at 62oC for 10 minutes after which 150 μl of 1.5% Triton-X was added and cells incubated for 15 minutes at 37oC while shaking at 900 rpm. 25 μl of 10X DpnII restriction buffer (NEB) was then added, and cells were incubated for 15 minutes while shaking. After that, 200U of DpnII (NEB R0543M) were added and incubated for 2 hours, then 200U more and incubated overnight. Next morning 200U more were added and incubated for 3h (total 600U of DpnII). DpnII was inactivated at 62oC for 20 minutes. For Hi-C, biotin fill-in was done by incubating cells with a mixture of 4.5 μl dCTP dTTP and dGTP at 3.3 mM, 8 μl klenow polymerase (NEB M0210L) and 37.5 μl Biotin-14-dATP (Thermo 19524016) for 4h at RT while shaking at 900 rpm for 10 seconds every 5 minutes. Ligation was done overnight at 16oC also rotating at 900 rpm for 10 seconds every 5 minutes by adding 120 μl of 10X ligation buffer (NEB), 664 μl water, 100 μl 10% Triton-X, 6 μl BSA 20mg/ml, and 2 μl T4 ligase (NEB cat #M0202M). For Capture-C, biotin fill-in step was skipped and 50 μl more of water was added to the ligation mix. Crosslink removal was done overnight with 50 μl of proteinase K in 300 μl of following buffer (10 mM Tris-HCl pH8.0, 0.5 M NaCl, 1%SDS) while shaking at 1400 rpm at 65oC. Following Sodium Acetate and 100% Ethanol -80oC precipitation, DNA was resuspended in 50 μl 10 mM Tris HCL.   Sonication was done using Covaris onetube-10 AFA strips using the following parameters for a 300 bp fragment size (Duration: 10 secs, repeat for 12 times, total time 120 secs, peak power-20W, duty factor 40%, CPB-50). Sonications were performed in a Covaris ME220 sonicator. Sonicated material was then size selected using SPRI beads with the following ratios: 0.55X and 0.75X. Hi-C material was then bound, washed and recovered to 150 μl Streptavidin C1 beads (Thermo 65002) per sample following manufacturers recommendations. Bead-bound DNA was resuspended in 50 μl 10 mM Tris HCl. Library preparation was done using Kapa Hyper Prep KK8502 kit. 10 μl of End-repair buffer and enzyme mix were added to resuspended beads and incubated for 30 minutes at RT and then 30 minutes at 65oC. 1 μl of 15 mM annealed-Illumina adaptors, containing an universal p5 and an indexed p7 oligo, were then incubated with a mixture containing 40 μl of ligase and ligation buffer at RT for 60 minutes. Libraries were then amplified using 4 reactions per sample for a total of 200 μl and 10 cycles, as recommended by manufacturer.