GSM6793471: AS14-03700, Control, scRNAseq; Homo sapiens; RNA-Seq
Thawed PBMCs were washed with PBS/0.04% BSA. Cells were counted and 100,000-1,000,000 cells were added to a 2 mL-microcentrifuge tube. Cells were centrifuged at 300xg for 5min at 4°C. The supernatant carefully completely removed, and 0.1X lysis buffer (1x: 10 mM Tris-HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2, nuclease-free H2O, 0.1% v/v NP-40, 0.1% v/v Tween-20, 0.01% v/v digitonin) was added. After 3 min incubation on ice, 1 ml of chilled wash buffer was added. The nuclei were pelted at 500 xg for 5 min at 4°C and resuspended in a chilled diluted nuclei buffer (10X Genomics) for scATAC-seq. Nuclei were counted and the concentration was adjusted to run the assay. ScRNAseq was performed as described (10x Genomics, Pleasanton, CA), following the Single Cell 3' Reagents Kits V3.1 User Guidelines. Cells were filtered, counted on a Countess instrument, and resuspended at a concentration of 1,000 cells/μl. The number of cells loaded on the chip was determined based on the 10X Genomics protocol. The 10X chip (Chromium Single Cell 3' Chip kit G PN-200177) was loaded to target 5,000-10,000 cells final. Reverse transcription was performed in the emulsion and cDNA was amplified following the Chromium protocol. Quality control and quantification of the amplified cDNA were assessed on a Bioanalyzer (High-Sensitivity DNA Bioanalyzer kit) and the library was constructed. Each library was tagged with a different index for multiplexing (Chromium i7 Multiplex Single Index Plate T Set A, PN-2000240) and quality controlled by Bioanalyzer prior to sequencing.