GSM8765539: nuRNA, DG, EE, rep3; Mus musculus; RNA-Seq
After tissue dissociation, nuclei were extracted by mechanical homogenization using a 2 ml Dounce homogenizer (Sigma-Aldrich) containing 500 μl of Nuclei Extraction Buffer (NEB; Sucrose 250 mM, KCl 25 mM, MgCl2 5 mM, HEPES-KOH pH 7.8 20 mM, IGEPAL CA-630 0.5 %, spermine 0.2 mM, spermidine 0.5 mM, and 1X proteinase inhibitors (cOmplete EDTA-free, Roche)). Tissues from 3 mice were pooled in each sample, having a total volume of 1.5 ml NEB containing the nuclei for each hippocampal layer. Samples were filtered in a 35 μm mesh capped tube and incubated with 0.01 mM DAPI for 10 min in darkness in a rotator. Nuclei isolation was accomplished by the preparation of a gradient of different densities in which nuclei stay in the interphase. Nuclei were diluted in Optiprep density gradient medium (1114542, Proteogenix) to a final concentration of 22 % Optiprep. For the density gradient, 44 % Optiprep was added to a centrifuge tube followed by 22 % Optiprep containing the nuclei. Additional 22 % Optiprep was added until the tube was filled. After centrifugation at 7500 rpm for 23 min, the phase containing the nuclei was collected in a new tube containing Nuclei Incubation Buffer (NIB: sucrose 340 mM, KCl 25 mM, MgCl2 5 mM, HEPES-KOH pH 7.8 20 mM, spermine 0.2 mM, spermidine 0.5 mM, 1X proteinase inhibitors, newborn calf serum 5 %). Sorting of SUN1-GFP+ nuclei was performed in a flow-cytometer FACS Aria III (BD Bioscience). Sorted nuclei were pelleted by centrifugation (1000 g, 7 min, 4 °C). RNA extraction was performed using TRI-reagent (Merck) as recommended by the manufacturer. After treatment with RNase-free DNase I (Qiagen) for 30 min at 25 °C to eliminate genomic DNA, RNA was precipitated with phenol-chloroform-isoamyl alcohol. RNA concentration was measured using NanoDropOne (Themo Scientific). RNA was retrotranscribed to cDNA using the RevertAid First-Strand cDNA Synthesis kit (Thermo Scientific) and analysed by RT-qPCR with PyroTaq EvaGreen (Cultek) in a QuantStudio 3 thermocycler (Applied Biosystems). TruSeq Stranded Total RNA, Illumina