GSM5583716: NOME-seq of Nng2 overexpressing cells rep2; Mus musculus; OTHER
The somatosensory cortex of in utero electroporated E14.5 mouse embryos was isolated and the electroporated area dissected. 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). Dissociated cells were fixed for 10 min. in 1% Formaldehyde (ThermoFisher, Cat. N: 28908), quenched by addition of Glycine (Invitrogen, Cat. N: 15527013) to a final concentration of 0.2M, washed in PBS with 0.1% BSA, passed through a 40µM cell strainer (ThermoFisher, Cat. N: 15342931) and immediately FAC-sorted at a BD FACSAria Fusion (BD Bioscience) with four lasers (405, 488, 561, 640) using a 100µm nozzle. Sorted cells were lysed with 0.2% Igepal-CA630 (Sigma-Aldrich, Cat. N: I3021) and incubated for 3h at 37˚C in a reaction mix containing 4.5U/µl M.CviPI (New England Biolabs, Cat. N.: M0227S) and 0.6mM SAM (New England Biolabs, Cat. N.: B9003). During the incubation the reaction was substitution with 4U M.CviPI and 0.5µl of 32mM SAM every hour. Thereafter nuclei were lysed and reverse-crosslinked overnight at 68˚C, purified by ethanol precipitation and sheared to ~550 bp fragments using a Covaris S220 sonicator. Methylated and unmethylated spike in controls were added to the samples (~ 0.05%) prior bisulfite conversion using the the EZ DNA Methylation-Gold kit (Zymo Research, Cat. N: D5005). Final libraries were constructed using the Accel-NGS® Methyl-Seq DNA Library kit (Swift Bioscience, Cat. N: 30024) according to the manufacturer's instructions until the final library amplification step. To increase the complexity of the samples, library amplification was performed in 5 separate reaction using the EpiMark Hot Start Taq (New England Biolabs, Cat. N: M0490S) with Methyl-Seq Indexing Primers (Swift Bioscience, Cat. N: 36024) using the following PCR program: 95˚C 30s; {95˚C 15s, 61˚C 30s, 68 ˚C 60s} x11; 68˚C 5min; Hold at 10˚C. Different PCR reactions of a sample were pooled and purified using 0.8x AMPure XP beads (Agencourt, Cat. N: A63881).