GSM8247656: PTx; Mus musculus; RNA-Seq
Mice were anesthetized with 87.5 mg per kg ketamine and 12.5 mg per kg xylazine and transcardially perfused with ice-cold phosphate-buffered saline. To process the brains, the whole brain was sectioned in half along the sagittal plane. The hippocampus from one hemisphere was subdissected and flash-frozen. For adoptive transfer with PTx, neuronal nuclei were isolated based on the demonstrated protocol by 10x Genomics with modifications and performed on nuclei isolated from three mice per group. Briefly, flash-frozen dissected hippocampi, three samples per group, were dounce homogenized (Wheaton, Cat# 357538) in 500μL of NP40 lysis buffer (10mM Tris-HCl (pH = 7.4), 10mM NaCl, 3mM MgCl2, 0.1% Nonidet P40 Substitute), 1mM DTT, 1x RNAse Inhibitor, Nuclease-free H2O) with 20 strokes of the loose pestle and 25 strokes of the tight pestle. 500 μL of NP40 lysis buffer was added and samples incubated for 7 minutes on ice. Samples were filtered through a 40 μm filter and centrifuged at 500 RCF for 5 min at 4°C. Samples were resuspended in 1ml of Wash Buffer (PBS, 1%BSA, 1xRNAse Inhibitor) and incubated for 5 min on ice. Samples were centrifuged at 500 RCF for 5 min at 4°C, supernatant was removed, and samples were resuspended in 400 uL of Wash Buffer with 1:10,000 dilution of Hoescht 33342 and incubated for five minutes before filtering through a 35μm FACS tube filter and sorting on a BD FACSAria II. Nuclei were sorted on a BD FACSAria Fusion with a 70 μm nozzle and with a flow rate of 1–2.5. Nuclei were first gated by forward and side scatter, then gated for doublets with height and width. Nuclei that were Hoechst+ were sorted and samples were combined per group. Samples were delivered to the UCSF-IHG Genomics Core for analysis with the 10x Genomics Chromium Single Cell Expression Solution 3′ kit with Feature Barcode Technology (v.3.1). The Genomics Core prepared cells for 10x Genomics Chromium single-cell capture. 30,000 nuclei were loaded per sample. cDNA libraries were prepared according to the standard 10x Genomics protocols. The final library pool was sequenced on the NovaSeq X 10B system at the UCSF CAT Core.