title: GSM8196003: sublibrary 10, batch 2, scRNAseq; Mus musculus; RNA-Seq
library_construction_protocol: To isolate nuclei from brain tissue, the right cortical hemisphere was dissected and homogenized. Prior to tissue homogenization, the following buffers were made: NIM1, homogenization, and resuspension buffers. NIM1 buffer was made by mixing 1190 μl of distilled water, 250 μl of 1.5M sucrose, 37.5 μl of 1M KCL, 7.5 μl of 1M MgCl2, 15 μl of 1M Tris Buffer, pH 8.0. Homogenization buffer was made by mixing 960 μl of NIM1 buffer, 1 μl of 1M DTT (Thermo Fisher Scientific), 10 μl of 40 U/μl RNase inhibitor (Enzymatics), 10 μl of 20 U/μl SUPERase-in RNase inhibitor (Thermo Fisher Scientific), 10 μl of 20 mg/ml BSA and 10 μl of 10% Triton X-100. Resuspension buffer was made by mixing 990 μl of PBS, 1.25 μl of 40 U/μl RNase inhibitor (Enzymatics), 2.5 μl of 20 U/μl SUPERase-in RNase inhibitor (Thermo Fisher Scientific), 10 μl of 20 mg/ml BSA and 5 μl of 10% Tween. Tenbroeck tissue homogenizer (Bellco Glass) was cleaned with distilled water, 100% ethanol and then distilled water and submerged in homogenization buffer. The tissue was homogenized using Tenbroeck tissue homogenizer and further homogenized by pipetting up and down 10-15 times using wide-bore pipette tips. Lysate (1.0 mL) was transferred into a DNA LoBind 1.5 mL tube (Eppendorf) and mixed. Homogenates was passively filtered with a 40 μm strainer (Falcon) into a 15 mL tube and then centrifuged at 500 x g for 5 minutes at 4 °C. The supernatant was carefully aspirated and the nuclei were resuspended gently with wide-bore tip in 1.0 mL cold resuspension buffer. After centrifugation and aspiration of the supernatant, the nuclei suspension was passively filtered again with a 40 μm strainer into a 15 ml tube. The nuclei suspension was fixed on ice for 10 minutes by slowly adding 3.0 ml of 1.33% formaldehyde. After 10 minutes, 20 μl of 10% Tween was added to the suspension and centrifuged at 500 x g for 5 minutes at 4 °C. The supernatant was carefully aspirated and the nuclei were resuspended in 1.8 mL cold resuspension buffer. After counting the nuclei, 200 μl of DMSO was added to the suspension and the isolated nuclei suspension was stored in -80°C until the library preparation step. Libraries from nuclei were prepared using the Split Pool Ligation-based Transcriptome sequencing (SPLiT-seq) method which utilizes a combinatorial barcoding technique to identify the cellular origin of RNA during snRNAseq analysis. 7,000 nuclei from each sample were used for library preparation. Cortical samples from a total of 92 CC mice were successfully processed. All libraries were pooled and sequenced on an Illumina NovaSeq 6000-S4.