GSM8827051: PIP-seq, postnatal day 14, replicate 3; Mus musculus; RNA-Seq
Hearts were harvested on postnatal day 14. Hearts were perfused with KCl and PBS before harvesting. After removing the heart from the chest, the ventricles of the harvested heart were minced into pieces on a glass petri dish on ice using a razor blade. A cut/bore tip was used to transfer tissue pieces from the glass petri dish to a 15ml falcon tube. The tube was then topped with fixation buffer (4% formaldehyde, 1X Fix & Perm Buffer) to reach a 25 mg tissue to 1ml fixation ratio. The tissue pieces were then incubated in the fixation buffer for 20 hours at 4°C without rotation. The incubation time should be consistent between samples for the same batch of experiments. After incubation, tissue pieces were centrifuged at 850 g for 5 min at room temperature. The supernatant was removed without disturbing the tissue pellet. Then the tissue pieces were washed again with 2 ml chilled PBS and resuspended in tissue homogenization buffer for nuclei isolation. The nuclei isolation for fixed heart tissue is almost the same as previously described for snRNA-seq. One notable difference is instead of performing 30 strokes with tight pestles, 50 strokes were performed for each heart. After nuclei isolation, flow cytometry was performed to enrich the GFP+/DRAQ+ population. Then, fixed RNA single-nucleus libraries were generated with the GFP+/DRAQ5+ population by strictly following the manufacturer's manual (Chromium Fixed RNA Profiling Reagent Kits for Singleplexed Samples, CG000691). During the spike in step of the custom probes, the left hand side probes were spiked at 20nM/probe instead of default 2nM/probe, and the right hand side probes were spiked at 10nM/probe instead of 2nM/probe as default.