title: GSM8647184: Cas9,NTC,day7, scRNA-seq; Homo sapiens; RNA-Seq
library_construction_protocol: Cryopreserved PBMCs were then retrieved from a liquid nitrogen tank, thawed in a 37°C water bath, and transferred into the cabinet after removing any remaining ice clumps. The outer surface of the container was sterilized with 5% ethanol, dried, and transferred into the cabinet. The cells were then transferred into 9 mL of T cell medium and centrifuged at 400g for 5 minutes at room temperature. After discarding the supernatant, the cells were resuspended in 5 mL of medium and counted using a ThermoFisher Attune NxT flow cytometer. Subsequently, the cells were seeded at a density of 1e6 cells/mL in plates or culture flasks. After 6 hours of PBMC revival, CD3/28 dynabeads were added for activation. Following 3 days of cell activation, the activation beads were removed using a magnet and the culture was continued for 24 hours. For electroporation, 24-well plates were prepared by adding 900 μL of PBMC complete medium per well and incubating at 37°C for 10 minutes. The Lonza 4D Nucleofector System with the P3 Primary Cell 4D kit was used, following the kit instructions for electroporation. Each electroporation reaction consisted of 1E6 cells, 1.2 pmol of EPIREG mRNA, and 40 pmol of sgRNA for each target gene. Immediately after electroporation, 100 μL of cell-free medium was added to each well, followed by a 1-hour incubation at 37°C. The cells were then transferred to 24-well plates for further culture. The cell suspension was loaded into Chromium microfluidic chips with 3' (v2 or v3, depending on the project) chemistry and barcoded with a 10× Chromium Controller (10X Genomics). RNA from the barcoded cells was subsequently reverse-transcribed and sequencing libraries were constructed using reagents from a Chromium Single Cell 3' v2 (v2 or v3, depending on the project) reagent kit (10X Genomics) according to the manufacturer's instructions. Sequencing was performed with Illumina (HiSeq 2000 or NovaSeq, depending on the project) according to the manufacturer's instructions (Illumina). The same library construction process was used in Huh7 cells.