Instrument: Illumina HiSeq 2500
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: SINGLE
Construction protocol: Mice were sacrificed by an overdose of Isoflurane or Ketamin/Xylazine, followed by perfusion through the left ventricle with artificial cerebrospinal fluid, equilibrated in 95%O2/5%CO2 on ice or 4°C. The dentate gyrus was then microdissected from 300 μ m vibratome brain sections. Single-cell suspensions were prepared using the Papain kit (Worthington) with 25-35min enzymatic digestion followed by manual trituration using a BSA-coated p1000 pipette or fire-polished Pasteur pipettes. Sampling was carried out on two experimental days, each day pooling from male and female mice in a single experiment. The dentate gyrus of male (day 1 p35, day 2 p24) and female (day 1 p12, day 2 p16) mice were dissected and dissociated separately. Suspensions were pooled at equal ratios. 1000 cells/μl were resuspended in artificial cerebrospinal fluid and added to the Reverse transcription mix to aim for sampling of around 3500 cells. All downstream cDNA synthesis (13 PCR cycles), library preparation and sequencing were carried out as instructed by the manufacturer (10X Genomics Chromium Single Cell Kit Version 1).