GSM9736896: GC, 7m, WT, nRNAseq, rep3; Mus musculus; RNA-Seq
Mouse cerebella were homogenized in ice-cold homogenization buffer (0.25 M sucrose, 150 mM KCl, 5 mM MgCl2, 20 mM Tricine-KOH pH 7.8, 0.15 mM spermine, 0.5 mM spermidine, 1 mM DTT, protease and phosphatase inhibitors, 20 U/ml SUPERase-In RNase inhibitor and 40 U/ml RNasin ribonuclease inhibitor) by 30 strokes with loose (A) pestle followed by 30 strokes with tight (B) pestle in a glass Dounce homogenizer on ice. The homogenate was supplemented with 0.92 volume of iodixanol solution (50% Iodixanol/Optiprep, 150 mM KCl, 5 mM MgCl2, 20 mM Tricine, pH 7.8, 0.15 mM spermine, 0.5 mM spermidine, 1 mM DTT, and protease and phosphatase inhibitors) and laid on a 27% iodixanol cushion. Nuclei were pelleted by centrifugation for 30 min at 17,200g at 4 °C in a standard tabletop centrifuge. The pellet was resuspended in the homogenization buffer and passed through a cell strainer. Nuclei were fixed with 1% formaldehyde for 7 min at room temperature and quenched with 0.125M glycine for 5 min at room temperature, followed by washes in homogenization buffer and wash buffer. Nuclei were blocked with block buffer (6% BSA, 0.05% Triton X-100 in PBS) for 1h at room temperature and incubated with anti-NeuN antibody (Millipore #MAB377, RRID:AB_2298772, 1:500-1:1000), then washed three times with wash buffer for 5 min, incubated with secondary antibodies (Invitrogen Alexa Fluor 488 Donkey anti-Mouse IgG #A21202, RRID: AB_141607, 1:500) for 30 min at room temperature, and washed three times with wash buffer. Labeled nuclei were resuspended in 1% BSA, 10 mM HEPES in PBS containing 5 ng/mL DAPI. Singlets were gated using DAPI, followed by the sorting of the NeuN+ population using a SONY MA900 Cell Sorter. RNA from sorted nuclei was purified using the AllPrep DNA/RNA FFPE Kit as in (Pressl et al., 2024; Xu et al., 2018). RNAseq libraries were prepared using the NEBNext Ultra II RNA library preparation kit for Illumina in conjunction with NEBNext multiplex oligos for Illumina. The quality of the sequencing libraries was evaluated using the Agilent 2200 TapeStation with D1000 High-Sensitivity ScreenTape. The samples were sequenced at the Rockefeller University Genomics Resource Center using a NextSeq 2000 sequencer to obtain 100-bp single-end reads.