GSM9136270: anti-Drebrin+_2; Homo sapiens; RNA-Seq
Nuclei were extracted in lysis buffer (nuclei extraction buffer (Miltenyi Biotex) with RNase inhibitor (Promega, N2615)) using a gentleMACS dissociator (Miltenyi Biotec). The nuclei suspension was then filtered through a MACS SmartStrainer (70 µm) with the strainer rinsed with lysis buffer. The filtered suspension was centrifuged at 300 x g for 5 min at 4 °C. The pellet was resuspended in 3 ml resuspension buffer (PBS + 0.04 % BSA + 0.2U/ul RNAse inhibitor), and filtered through a MACS SmartStrainer (30 µm). The collected nuclei suspension was immediately processed for debris removal using density gradient centrifugation. For debris removal, the nuclei suspension was centrifuged at 300 x g for 10 min at 4 °C, resuspended in 900 µl PBS, and gently layered with 355 µl debris removal solution (Milteny), followed by 4 ml PBS. Density gradient centrifugation was performed at 3000 x g for 10 min at 4 °C. The two top phases were aspirated, and the tube was filled up with 15 ml PBS, inverted to mix, and centrifuged again at 1000 x g for 10 min at 4°C. The final nuclei pellet was resuspended in 500 µl resuspension buffer and stained with DAPI (1.7µg/ml, Sigma-Aldrich). Nuclei were sorted using BD FACSAria III (100 µm nozzle size, 20psi, 4°C). Nuclei were prepared using the dual-index Chromium Next GEM Single Cell 3' Kit v3.1 (10xGenomics) with a target nuclei load of approximately 16,000 per sample. Sequencing was performed on a Novaseq6000 (28/8/91, Illumina) using S2 flow cell with a target of 50,000 reads per nucleus following standard protocols.