GSM6165793: P26, snrna, rep1; Homo sapiens; RNA-Seq
Fresh blood samples were kept on ice until isolation was started. In a sterile laminar flow hood, equal parts blood and Dulbecco's phosphate-buffered saline (PBS) were mixed with 2% fetal bovine serum (FBS; STEMCELL Technologies, Cat. #07905). CCardiac nuclei were isolated by using density gradient centrifugation with Optiprep Density Gradient Medium (Sigma). All nuclei were isolated from the 30% to 40% interface and then diluted into nuclei wash buffer (PBS containing 1.0 % bovine serum albumin [BSA]) (Sigma) with 0.2 U/uL RNase inhibitor (Enzymatics/Qiagen, Y9240L) before being spun down at 1000 x g for 5 minutes. Next, the supernatant was removed, and the nuclei were washed twice with 25 mL of nuclei wash buffer. Nuclei were then resuspended in an appropriate volume of nuclei wash buffer to achieve a concentration appropriate for use with 10X Genomics Single Cell 3' Reagents. LymphoprepTM solution (STEMCELL Technologies, Cat. #07801) was added to a SepMateTM-15 (IVD) tube (STEMCELL Technologies, Cat. #85415). The blood was carefully layered on top of the LymoprepTM solution and spun at 1200 G for 20 minutes. The plasma layer was pipetted off, aliquotted, and stored at –80°C. The PBMC layer was poured off and washed in 10 mL of Dulbecco's PBS with 2% FBS and spun at 1000 G for 4 minutes to pellet PBMCs for downstream experiments. For PBMCS, the single cell 3' v2 protocol (10x Genomics) was used. For cardiac nuclei, the single cell 3' v3 protocol (10x Genomics) was used. Library preparaation was performed according to the manufacter's instructions.