title: GSM8318246: Panc-1, AA, rep2; Homo sapiens; RNA-Seq
library_construction_protocol: Nuclei were isolated from Panc-1 cells using an IgePal-based lysis buffer (protocol optimized from (Krishnaswami et al, 2016)) , see Supplemental Material for further details). Briefly, 4x106 cells were collected in BSA-coated DNA LoBind tubes (Eppendorf Cat. #022431021), lysed over ice (10 min) and centrifuged (350 RCF, 5 min, 4°C). Lysed cells/nuclei were washed in ice-cold PBS supplemented with BSA (3%), Protease and RNAse inhibitors, followed by centrifugation (800 RCF, 10 min, 4°C). This wash step was repeated twice. For optimal removal of cell membrane, nuclei were spun down through a gradient of Iodixanol (50 % over 29%, 14000 RCF, 22 min, “Optiprep”, Stemcell Cat #7820), washed and filtered through 30 µm filters (Miltenyi Biotec Cat. #130-098-458). Nuclei isolation was verified by BF imaging, and a control sample was stained with 4',6-diamidino-2-phenylindole (DAPI) to assess nuclear membrane integrity. Nuclei were counted and adjusted to a final concentration of 700-1200 nuclei/µL in nuclei storage buffer. cDNA libraries were prepared using 10X Genomics Chromium technology. Briefly, for each sample, 5-10*104 nuclei along with reagents, and partitioning oil were loaded onto the 10X Chromium chip (3' V3.1) according to manufacturer's recommendations (Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1, Document Number CG000204 Rev D, 10x Genomics), and processed in the 10X Chromium controller (10X Genomics). Indexed libraries were pooled, then sequenced in two lanes.