GSM4710402: MethylHiC E14.5 Eomes+ rep3; Mus musculus; OTHER
The somatosensory cortex of E14.5 mouse embryos was isolated and meninges removed. Single cell suspension was prepared using the papain-based neural dissociation kit (Miltenyi Biotec, Cat. N: 130-092-628) according to the manufacturer protocol with reduced dissociation times (5 min. instead of 15 min. at 37˚C). Cell number as well as the viability was assessed using the Countess™ II Automated Cell Counter (Invitrogen). For ImmunoFACS the dissociated cells were fixed for 10min. at RT in 1% freshly prepared Formaldehyde in PBS (ThermoFisher, Cat. N: 28908) under slow rotation and quenched by addition of Glycine (Invitrogen, Cat. N: 15527013) to a final concentration of 0.2M. The fixated cells were spun down with 500g for 5min. at 4˚C, washed once with 1% BSA (ThermoFisher, Cat. N: AM2618) in PBS and subsequently incubated for 10min. at 4°C in permeabilization buffer consistent of 0.1% freshly prepared Saponin (Sigma-Aldrich, Cat. N: SAE0073), 0.2% BSA and 1% RNAsin plus RNase inhibitor (Promega, Cat. N: N261A) in PBS. The permeabilization buffer was removed by centrifuging the cells with 2500g for 5min. at 4˚C followed by staining against Pax6 (1:40, BD 561664), Eomes (1:33, BD 566749) and Tubb3 (1:14, BD 560394) in staining buffer (0.1% saponin, 1% BSA, 0.1% RNAsin plus RNase inhibitor in PBS) for 1h at 4˚C under slow rotation. Cells were washed twice with permeabilization buffer, once with PBS with DAPI (1:1000; ThermoFisher, Cat. N: 62248) and a final wash with PBS containing 0.5% BSA. Between each washing step, the cells were incubated for 5min. at 4˚C with the respected buffer under slow rotation and spun down with 2500g for 5min. at 4˚C. After the last wash the cells were resuspended in PBS with 1% BSA and 1% RNAsin plus RNase inhibitor, passed through a 40µM cell strainer (ThermoFisher, Cat. N: 15342931) and immediately FAC-sorted. For Methyl-Hi-C we adapted current protocols (Lee et al., 2019; Li et al., 2019). Briefly, frozen pellets of the fixed and sorted cells were thawed on ice, lysed with 0.2% Igepal-CA630 (Sigma-Aldrich, Cat. N: I3021), permeabilized with 0.5% SDS (Invitrogen, Cat. N:AM9823) and digested with 200U DpnII (New England Biolabs, Cat. N: R0543) overnight at 37˚C. Subsequently, sticky ends were filled by incubating the nuclei for 4h at RT with DNA Polymerase I (New England Biolabs, Cat. N: M0210) and a biotin-14-dATP (Life Technologies, Cat. N: 195245016) containing nucleotide mix in DpnII buffer followed by proximity ligation for at least 6h at 16 °C. Thereafter nuclei were lysed, proximity ligated DNA was reverse-crosslinked overnight at 68˚C followed by a purification by ethanol precipitation. DNA was sheared to ~550 bp fragments using a Covaris S220 sonicator and end-repaired by incubation the samples with the T4 DNA Polymerase (New England Biolabs, Cat. N: M0203) for 4h at 20˚C. Prior the bisulfite conversion, sheared and biotinylated fully methylated pUC19 (Zymo Research, Cat. N: D5017) and unmethylated lambda DNA (Promega, Cat. N: D1521) was added to the samples (~ 0.01%). Bisulfite conversion was performed using the EZ DNA Methylation-Gold Kit (Zymo Research, Cat. N: D5005) followed by library construction using the Accel-NGS® Methyl-Seq DNA Library Kit (Swift Bioscience, Cat. N: 30024) according to the manufactory instructions until the adapter ligation step. After this step, biotin pulldown was performed using MyOne Streptavidin T1 beads (ThermoFisher, Cat. N: 65602) followed by 5 washes with washing buffer containing 0.05% Tween-20 (Sigma-Aldrich, Cat. N: P9416) and two additional washes with low-TE water. Libraries were amplified on the streptavidin beads using the EpiMark Hot Start Taq (New England Biolabs, Cat. N: M0490) using following program: 95˚C 30s; {95˚C 15s, 61˚C 30s, 68 ˚C 60s} x14; 68˚C 5min; Hold at 10˚C. Streptavidin T1 beads were pelleted on a magnetic rack and the prepared libraries within the supernatant were purified using 0.6x AMPure XP beads (Agencourt, Cat. N: A63881) to reach an average fragment size of approximately 500bp. Before sequencing, libraries were quantified by qPCR using the NEBNext® Library Quant Kit (New England Biolabs, Cat. N: E7630S). The size distribution of the obtained libraries was assessed using Agilent 2100 Bioanalyzer.
GEO Accession
GSM4710402