Brains were extracted using the Adult mouse and rat brain dissociation kit and the gentleMACS Octo dissociator from Miltenyi biotech. Single-cell RNA-Seq libraries were generated from each time point using the 10X Genomics Chromium Controller Instrument (10X Genomics, Pleasanton, CA) and ChromiumTM Single Cell 3ʹ GEM, Library & Gel Bead Kit v3, 16 rxns PN-1000075, according to manufacturer's instructions. Reverse transcription and sample indexing was performed using the C1000 Touch Thermal cycler with 96-Deep Well Reaction Module. Briefly, the suspended cells were loaded on a Chromium controller Single-Cell Instrument to first generate single-cell Gel Bead-In-Emulsions (GEMs). After breaking the GEMs, the barcoded cDNA was then purified and amplified. The amplified barcoded cDNA was fragmented, A-tailed and ligated with adaptors. Finally, PCR amplification was performed to enable sample indexing and enrichment of the 3' RNA-Seq libraries. The final libraries were quantified using Thermo Fisher Qubit dsDNA HS Assay kit (Q32851) and the fragment size distribution of the libraries were determined using the Agilent 2100 BioAnalyzer High Sensitivity DNA kit (5067-4626). Pooled libraries were then sequenced using Illumina Sequencing By Synthesis (SBS) chemistry.
GEO Accession
GSM4709812