GSM8765287: hBMSC-TERT4 cells, Ob 4 hrs, rep 3; Homo sapiens; RNA-Seq
Bulk RNA was extracted from cultured cells using Trizol combined with Econo Spin columns (Epoch Life Sciences). For Parse snRNA-seq, cells have been trypsinized and spun down at 200 g cells. Supernatant was carefully removed without touching the upper layer. Cells were subjected to nuclei isolation following a protocol adjusted from Hauwaert et al. (STAR Protocols 2:100612). Briefly, cells were dounced 5 times in nuclei isolation buffer consisting of 250 mM Sucrose (84097, Sigma-Aldrich), 10 mM HEPES (22320022, Thermo Fisher Scientific), 1.5 mM MgCl2 (M2670, Sigma-Aldrich), 10 mM KCl (P9541, Sigma-Aldrich), 0.001% IGEPAL CA-630 (I8896, Sigma-Aldrich), 0.2 mM DL-Dithiothreitol (43819, Sigma-Aldrich) and 0.5 U/mL RNase inhibitor (40,000 U/mL, M0314L, Bionordika) in DEPC-treated water. Nuclei were passed through a 30 µm cell strainer, centrifugated at 200 g for 10 min at 4C and resuspended in nuclei resuspension buffer consisting of 1% BSA (A9418, Sigma-Aldrich) in PBS, 2 mM MgCl2 and 0.04 U/µL RNase inhibitor in PBS followed by a final transfer through a 30 µm cell strainer. Nuclei fixation was carried out according to manufacturer's instructions (Parse Biosciences Evercode WT, version 2). For 10x scRNA-seq, cells have been trypsinized and spun down at 200 g and washed twice in culture media. Cells were separated using a 40 µm mesh, washed 3 times in PBS with 0.04% non-acetylated BSA, and cell numbers were adjusted to 0.7 million cells per ml followed by a final transfer through a 40 µm mesh. RNA-seq was performed according to manufacturer's instructions (TruSeq 2, Illumina) using 1 µg RNA for preparation of cDNA libraries. scRNA-seq: Libraries were prepared according to manufactures instructions (10x Genomics). snRNA-seq: Libraries were prepared using a WTmini loading scheme according to manufactures instructions (Parse).