GSM9010865: Individual_snRNA-seq_Donor_6; Homo sapiens; RNA-Seq
Nuclei were isolated from heart tissue by mechanical disruption, followed by centrifugation through sucrose and purification from debris by FACS. In brief, frozen heart tissue (~100 mg) was suspended in 5 ml of lysis buffer and thawed on wet ice. When samples were pooled for 10x RNA-Seq (multiple human samples, or sheep and human), samples were either combined after (human real-world dataset) or prior to homogenization (human application dataset and sheep-human species-mixed dataset), where equivalent weights of each tissue sample were pooled. Next, samples were homogenized using a gentleMACS dissociator (Miltenyi) with gentleMACS M tubes and the Protein_01_01 protocol. 5 ml of lysis buffer containing NP-40 was added to the homogenate and incubated on wet ice for 15 min. Then, the suspension was filtered through a 30 μM cell strainer (Miltenyi). Residual nuclei were recovered by washing the M tube and filter with 2 ml of lysis buffer containing NP-40, which was then combined with the remainder of the homogenate. The homogenate was centrifuged in a swinging-bucket centrifuge (Eppendorf, 5920R) at 1000 g for 5 min (4 ℃, ramp speed 4/4). The supernatant was carefully removed and the pellet containing nuclei was resuspended in 2 ml of sucrose buffer, which was then layered on top of 2 ml of sucrose buffer. The sample was then centrifuged at 500 g for 5 min (Eppendorf, 5920R; 4 ℃, ramp speed of 4/4) to recover nuclei. The supernatant was removed and the pellet was resuspended in 500 μl of wash buffer and centrifuged at 500 g for 5 min (Eppendorf, 5920R; 4 ℃, ramp speed of 4/4). The supernatant was carefully removed and stained with 1 ml of staining buffer containing DAPI (1:1000; 7406). DAPI-positive nuclei were separated from cell debris by flow sorting using a SONY sorter instrument (SONY MA900) and collected into 50 μl of collection buffer. Sorted nuclei were then collected by centrifugation at 1000 g for 5 min (Eppendorf, 5920R; 4°C, ramp speed of 4/4), and the concentration was adjusted to 1,000 nuclei/µl targeted for predicted capture 10,000. Nuclear suspensions were loaded on a 10x Chromium Controller (10x Genomics) and processed with 10x Chromium Single Cell 3' Reagent Kit v3 (10x Genomics) according to the manufacturer's protocol. Cell-RT mix was prepared, targeting 10,000 cells per sample. Library quantification and quality assessment were performed using the Bioanalyzer Agilent 2100 with a High Sensitivity DNA chip (Agilent Genomics). Indexed libraries were pooled equimolarly and sequenced on an Illumina NovaSeq 6000 by GenomeScan using paired-end 26x98-bp reads as the sequencing mode. Libraries were sequenced on the NovaSeq 6000 (Illumina) at 30,000 reads per nuclei resolution.