GSM6186019: HiC-seq for E12-E15 brain cortex, rep1; Mus musculus; Hi-C
Cell suspension was prepared from E10 to E17 CD-1 mouse embryos. Four brains were collected for each time point. For E10 and E11 time points, ventricular zone was dissected from the tip of the brain. For E12 to E17 time points, brain tissues were first embedded in 4% low melting agarose at 35-37 °C and sectioned at 200 μm thickness with a vibratome (Leica) in ice-cold and oxygen-bubbling artificial cerebrospinal fluid (ACSF) containing: 125 mM NaCl, 5 mM KCl, 1.25 mM NaH2PO4, 1 mM MgSO4, 2 mM CaCl2, 25 mM NaHCO3, and 20 mM glucose (pH 7.4). The brain sections were transferred to a 3.5 cm dish containing ice-cold hibernate-E medium, and then ventricular zone was dissected and picked up under a stereoscope. The isolated VZ tissues were digested with papain using a commercial kit (Worthington, LK003150) at 37 °C for 30 min according to the manufacturer's recommendations. Single cell suspension was generated by dissociating the digested tissues through pipetting, washed with DPBS to remove digesting enzymes, and stained with DAPI in ice-cold DPBS. To enrich RGPs at later time points, FlashTag labeling was performed as previously described (Govindan et al., 2018). Briefly, pregnant mice were anesthetized, injected with FlashTag into the lateral ventricle of the uterine horns, and housed for 30 min. Then, single cell suspensions were prepared as described above. Hi-C libraries were prepared as described previously (Díaz, N. et al. Nat Commun. 2018) with minor modifications: 1x105 FlashTag+ FACS-purified cortex cells were immediately fixed for 10 min in freshly prepared 1% formaldehyde (Thermo Fisher Scientific, 28906) in PBS (Thermo Fisher Scientific, AM9624) at room temperature with gentle rotation (300 rpm). Then the reaction was quenched in 0.2 M glycine (Sigma-Aldrich, 50046) solution for 5 min at room temperature with gentle rotation (300 rpm). The cells were washed twice with 1 ml cold 1x PBS (centrifuged at 400 g for 5 min at 4°C). To lyse cells, the cells were resuspended in ice-cold in situ Hi-C buffer and incubated on ice for 15 min. The lysed cells were frozen in liquid nitrogen and stored at -80 °C until further use. Chromatin was digested using 50 U of MboI (New England Biolabs, R0147S) overnight at 37 ℃ with gentle rotation. After biotin filling, proximity ligation was carried out using 17.5 U T4 DNA Ligase (Thermo Fisher Scientific, EL0012) for 4 h at 20 °C with gentle rotation. DNA was sheared to 200-500 bp fragments using Covaris S220 sonicator with a duty factor of 10%, peak power of 140 W, and 200 cycles per burst. Each sample was sonicated for 2 cycles, 50s/cycle. Ligation fragments containing biotin were immobilized on MyOne Streptavidin C1 beads (Thermo Fisher Scientific, 65001). Libraries were prepared using NEBNext® Ultra™ II DNA Library Prep Kit, according to the manufacturer's instructions. Libraries were PCR amplified for 14-16 cycles by using NEBNext® Multiplex Oligos. DNA was then purified using 2.2x VAHTS DNA Clean Beads and eluted in ultrapure H2O.