GSM9012137: Control_1; Mus musculus; RNA-Seq
The medial neocortices from E16.5 Sst-Cre/tdTomato and Sst-Cre/tdTomato/Dnmt1 loxP2 mice were dissected. The samples were snap-frozen on dry ice and stored at -80 °C until library preparation to ensure RNA integrity was maintained while the genotype and the sex of each sample could be verified for a bias-free transcriptomic analysis. Consequently, single-nucleus RNA sequencing (snRNA-seq) was selected over scRNA-seq to accommodate the processing of frozen samples. The nuclei were extracted from the frozen tissue by discontinuous sucrose gradient ultracentrifugation. Briefly, the tissue was homogenized in 1 ml of lysis buffer (0.32 M sucrose, 10 mM Tris-HCl (pH 8.0), 5 mM CaCl2, 3 mM Mg acetate, 1 mM dithiothreitol (DTT), 0.1 mM EDTA, 0.1% Triton X-100, 50 U/ml RNase inhibitors (#M0314S, New England Biolabs, U.S.A.)) using a 2-ml glass Dounce homogenizer on ice and stroking 25 times with pestle A followed by 100 strokes with pestle B. The homogenates were transferred to 5-ml ultracentrifuge tubes (#344057, Beckman Coulter) and under-layered with 3 ml of sucrose cushion (1.8 M sucrose, 10 mM Tris-HCl (pH 8.0), 3 mM Mg acetate, 1 mM DTT, 50 U/ml RNase inhibitors). The samples were loaded onto a Sw 55 Ti rotor and centrifuged for 1 h at 4 °C and 28,300 rpm (97,500x g). The nuclei pellets were resuspended in 150 µl of cold 1% BSA in PBS and immediately fixed using the Evercode™ Nuclei Fixation kit (Parse Biosciences, U.S.A.) following the manufacturer's guidelines. The fixed nuclei were stored at  80 °C until library preparation. The snRNA-seq libraries were generated with the Evercode™ WT kit (Parse Biosciences, U.S.A.). The quality check of the cDNA and the final libraries was performed using the High Sensitivity D5000 and D1000 ScreenTape kits (Agilent Technologies, U.S.A.). Libraries were equimolarly pooled and sequenced in conjunction with 5% PhiX control library on an Illumina NovaSeq machine in paired-end mode using the S2 Reagent Kit v1.5 (200 cycles) (Illumina, U.S.A.).