GSM9707898: PKP2fl/fl MCM cre/+ mice R1; Mus musculus; RNA-Seq
Frozen tissue punches were transferred to a prechilled Dounce homogenizer tube, followed by the addition of ice-cold detergent lysis buffer (composition: 0.32 M sucrose, 10 mM HEPES [pH 8.0], 5 mM CaCl₂, 3 mM MgAc, 0.1 mM EDTA, 1 mM DTT, 0.1% Triton X-100). Tissue lysis and nucleus release were performed by homogenization (10 strokes with pestle A, followed by 10 strokes with pestle B). &#10;The lysate was then diluted with chilled low-sucrose buffer (composition: 0.32 M sucrose, 10 mM HEPES [pH 8.0], 5 mM CaCl₂, 3 mM MgAc, 0.1 mM EDTA, 1 mM DTT) and filtered through a 40 μm cell strainer to remove cellular debris. The filtered suspension was centrifuged at 3,200 × g for 10 minutes at 4 °C, and the supernatant was carefully decanted. &#10;The nuclear pellet from each sample was resuspended in 750 μL of chilled resuspension buffer (1× PBS, 0.4 mg/mL BSA, 0.2 U/μL RNase Inhibitor [Biosearch Technologies, Cat. 30281]) containing one of eight unique XPoSE-tags. Each XPoSE-tag consists of a Nucleoporin 62 antibody conjugated to R718 fluorescent dye and one of eight distinct oligo-based Sample Tags (STs), which are custom reagents (BD Biosciences) used at a 1:2000 dilution to uniquely barcode nuclei from individual samples. &#10;For verification of the neuronal nuclei gate, both the XPoSE-tag and the neuronal nuclear marker NeuN (anti-NeuN Mouse mAb, clone A60, MAB377X, Millipore Sigma, RRID: AB_2149209) were added at a 1:500 dilution. Samples were incubated with the antibodies for 15 minutes at 4 °C on a rotating mixer, then transferred to a 5 mL polystyrene round-bottom tube (Falcon) containing 3.5 mL of chilled resuspension buffer. &#10;The samples were centrifuged at 250 × g for 10 minutes at 4 °C, and 4 mL of the supernatant (containing residual XPoSE-tag) was carefully aspirated. The pellet containing XPoSE-tag-labeled nuclei was resuspended in 1–2 mL of chilled resuspension buffer and filtered through a 40 μm cell strainer prior to fluorescence-activated nuclei sorting (FANS). Single-cell whole transcriptome mRNA and Sample Tag libraries (BD Biosciences, cat. 633801) were generated according to the manufacturer's protocols for sequencing on Illumina® sequencers. Library concentration was measured using a Qubit 4 fluorometer (ThermoFisher, cat. Q33230) and length distribution via a Bioanalyzer 2100 (Agilent, cat. 5067-4626), following the respective manufacturer's protocols. Both cartridges were processed in parallel to generate indexed, separate libraries, which were pooled for sequencing. Whole transcriptome libraries were sequenced at 45,000 reads per nucleus and Sample Tag libraries at 1,000 reads per nucleus on an Illumina Novaseq S4 lane (PE 150 bp).