GSM4907713: Control2; Homo sapiens; RNA-Seq
Five 10 µm curls cut from OCT embedded human cardiac tissue were homogenized using a 2 ml Dounce homogenizer with 1 ml chilled lysis buffer (10mM Tris-HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl2, 0.1% NP-40 and 0.2 U/ml RNase inhibitor). After filtering through a 40 µm cell trainer, nuclei were centrifuged at 700 x g for 5 min at 4oC. The nuclei were subsequently washed with 500 ml and then resuspended in 300 ml of Nuclei Wash/Resuspension Buffer (1x PBS with 2% BSA and 0.2 U/ml RNase inhibitor). 1 ml DRAQ5 (5 mM solution, Thermo Cat #62251) was added, and DRAQ5+ nuclei were sorted (70 mm nozzle on a BD Aria) into 50 ml Nuclei Wash Buffer (3 X 0.2 U/ml RNase inhibitor). Sorted nuclei were directly added to a reverse transcription master mix diluted with water to manufacturer's specifications Libraries were prepared using the 10X Chromium Single Cell 3' v3 Gene Expression Kit.