GSM9158951: E16 miR-9-2 Wild-type male 2 cerebral cortex; Mus musculus; RNA-Seq
For nuclei isolation, samples were processed via a modified 10X Genomics Nuclei Isolation from Mouse Brain Tissue protocol (CG000212 Rev. B). 500 µl of cold 0.1X Lysis Buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.01% Tween-20, 0.01% IGEPAL CA-360, 1% BSA, 1 U/µl RNase Inhibitor (Roche #03335402001)) was added to each sample-containing tube and triturated 15-20 times. The suspensions were further homogenized using dounce homogenizers by applying 10 dounces each with an A then B pestle. Following transfer of each sample to a new tube, the samples were incubated on ice for 5 minutes. Sample suspensions were pipette-mixed 10 times, then incubated on ice for 10 minutes. 500 µl of cold Wash Buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 1% BSA, 0.1% Tween-20, 1 U/µl RNase Inhibitor) was then added, and samples were pipette-mixed 5 times and then passed through a 40 µm Flowmi Cell Strainer into a fresh tube. Dissociated sample nuclei were then washed 2 times by centrifugation pelleting at 500 rcf in a 4ºC swinging-bucket centrifuge and resuspension in 750 µl cold Wash Buffer. Sample nuclei concentrations were determined using propidium iodide and a Countess II FL Automated Cell Counter. Nuclei were then centrifuged at 500 rcf for 5 minutes at 4ºC. The supernatant was discarded and the nuclei were resuspended in cold Nuclei Buffer (1X 10X Genomics Nuclei Buffer) to achieve a nuclei concentration range of 700-1200 nuclei/ µl, which is recommended for targeting 10,000 cells in the 10x Genomics snRNA-seq protocol. Sample libraries were generated following the 10X Genomics Chromium Next GEM Single Cell 3' v3.1 snRNA-seq protocol and subsequently sequenced per 10X Genomics' recommended cycles and sequencing depth using either a NovaSeq SP or NovaSeq S1 100-cycle flow cell, according to the number of samples submitted for sequencing (SP for 4 or S1 for 5-9 samples).