GSM8101991: Batch2 - HTO; Mus musculus; RNA-Seq
The top left lobe of the liver was removed and flash-frozen in liquid nitrogen and stored at −80°C until sample processing. Single-nucleus pools from frozen livers were generated with several modifications. Briefly, frozen tissue was dounce homogenized with 10 strokes of the loose pestle followed by 10 strokes with the tight pestle at 4 °C in a low sucrose buffer (0.32 M sucrose, 10 mM HEPES (pH 7.5), 5 mM CaCl2, 0.1 mM EDTA, 3 mM MgAc, 1 mM DTT) with 0.1% Triton X-100 added. The crude nuclei prep was gently mixed with additional low sucrose buffer and was subsequently passed over a pre-wet 30 μm strainer. The filtered crude nuclei prep was centrifuged at 2613 × g for 10 min at 4°C. Supernatant was decanted and the pellet was re-suspended in low sucrose buffer. An additional homogenization step using a TissueRuptor II (Qiagen Inc.) set to the lowest power was completed on each sample for 15 s. High sucrose buffer (1 M sucrose, 10 mM HEPES (pH 7.5), 3 mM MgAc, 1 mM DTT) was then layered underneath the crude prep. The crude prep was centrifuged for 25 min at 2613 × g at 4°C. The supernatant was decanted and re-suspended in resuspension buffer, 1% BSA in PBS with 0.2 U/μL of Ambion RNase Inhibitor (Invitrogen, Cat:AM2684), and transferred to a low-bind conical tube. The nuclei were washed of any residual sucrose by centrifugation at 500 × g for 5 min at 4°C. The pellet was re-suspended in PBS containing 20 μg/mL Fc Block CD16/CD32 (Biolegend, Cat: 101301) and incubated for 5 min at 4 °C. 0.5 μg of a nuclear hashing antibody (10 μg/ml) was added to each sample and incubated for 30 min at 4 °C. Nuclei were washed three times and finally counted using a Countess Automated Cell Counter (ThermoFisher). Two pools of nuclei were generated each containing 40,000 nuclei from samples of the four experimental groups. Nuclear hashtag antibodies used included anti-Nuclear Pore Complex Proteins Hashtag Antibodies 1–4 (BioLegend, Catalog numbers: TotalSeq-A0451, TotalSeq-A0452, TotalSeq-A0453, TotalSeq-A0454). The 10X Chromium controller (10X Genomics) with v3 chemistry was used to capture, barcode and prepare single-nucleus libraries from each pool of nuclei as per manufacturer's instructions. The resulting libraries were sequenced on a full lane of an Illumina NovaSeq 6000 S2 flow cell for average depths of 9–10 k reads per nucleus depending on the 10X batch.