title: GSM8703567: 01WTM_MB21_TAO574_posM_scRNA_2M216_BDS_S1_DGE; Mus musculus; RNA-Seq
library_construction_protocol: RNA was extracted from cortical tissue using the RNeasy Universal Kit (Qiagen) following established protocols. Quality control criteria for RNA samples included an RNA integrity value above 8.5 and absorbance ratios at 260/230 and 260/280 equal to or greater than 1.8 and 2, respectively. Validated samples were stored at -80°C. For single-cell transcriptomics, fresh cortical tissue was dissociated using the Adult Brain Dissociation kit (Miltenyi Biotec) before proceeding with DropSeq analysis. The 3′ end enriched cDNA libraries were prepared by tagmentation reaction of 600 pg cDNA library using the standard Nextera XT tagmentation kit (Illumina). Reactions were performed according to the manufacturer's instructions. The PCR amplification cycling programme used was 95 °C 30 s, and 12 cycles of 95 °C (10 s), 55 °C (30 s), and 72 °C (30 s), followed by a final extension step of 72 °C (5 min). Libraries were purified twice to reduce primers and short DNA fragments with 0.6 × and 1 × Agencourt AMPure XP beads (Beckman Coulter), respectively, in accordance with the manufacturer's protocol. Finally, purified libraries were eluted in 10 μl Molecular Grade Water. Quality and quantity of the tagmented cDNA library were evaluated using Bioanalyzer High Sensitivity DNA Chip. The average size of the tagmented libraries prior to sequencing was between 400 and 700 bps. Purified DropSeq libraries were sequenced on an Illumina NextSeq machine using paired-end sequencing. Read 1 (20 bases) covered cell barcodes (bases 1-12) and unique molecular identifiers (UMIs, bases 13-20), while Read 2 captured 60 bases of gene sequences. A custom primer (GCCTGTCCGCGGAAGCAGTGGTATCAACGCAGAGTAC) was used for Read 1 at 6pM concentration.