GSM8187204: Multiome - GEX - Sample 1; Homo sapiens; OTHER
Co-cultured MM1S and HS5 cells were processed for single-cell Multiome ATAC + Gene Expression (10X Genomics) with a targeted nuclei recovery of approximately 10,000. Nuclei were isolated according to the Demonstrated Protocol: Nuclei Isolation for Single Cell Multiome ATAC + Gene Expression (10X Genomics, CG000365 Rev A). Approximately one million cells were added to a 5.0 mL low binding tube and centrifuged (300×g for 5 min at 4°C) using a swinging bucket rotor. Cells were washed twice with PBS +0 .04% BSA and were passed through 40uM Flowmi cell strainer to remove any clumps. After pelleting the strained cells, the cell pellet was resuspended in 100 µL of chilled 10X Genomics Lysis Buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20, 0.1 % NP-40 Substitute, 0.01% digitonin, 1% BSA, 1 mM DTT, 1 U/μL RNase inhibitor 40 U/mL) by pipette-mixing 10 times. Cells were incubated on ice for 3 min, followed by dilution with 1 mL of chilled Wash Buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20, 1% BSA, 1 mM DTT, 1 U/mL RNase inhibitor 40 U/mL). Nuclei were then centrifuged (500×g for 3 min at 4°C), and the supernatant was slowly removed. The nuclei were washed one additional time with 1 mL Wash Buffer. Nuclei were resuspended in chilled diluted nuclei buffer (1X Nuclei Buffer, 1 mM DTT, 1 U/mL RNase inhibitor 40 U/mL). The single-cell ATAC library construction and gene expressionlibrary construction was carried out as described in the Chromium NextGEMSingle CellMultiomeATAC +Gene Expression User Guide (CG000338 Rev A). ATAC and GEX libraries weresequenced separately on anHiSeq4000(Illumina) before demultiplexing,alignment to the reference genome, andpost-alignment quality control.