title: GSM6170854: Replicate 4 – IFNa - 24h; Homo sapiens; RNA-Seq
library_construction_protocol: Cells were filtered using a 30 µm cell strainer (Corning) and resuspended in 200 µL Dead Cell Removal MicroBeads (Dead Cell Removal kit, Miltenyi Biotec). The dead cells were removed according to the manufacturer's instructions, using LS columns and MidiMACS separator (Miltenyi Biotec). The final cell suspension was centrifuged at 300 rcf for 3 min and resuspended into 1 ml of PBS + 5% BSA. The cell count and the cell viability were determined using a Countess II Automated Cell Counter and trypan blue solution (Thermo Fisher Scientific). The single cell gene expression analysis was conducted using a 10X Chromium single cell system (10X Genomics, Inc) and a NovaSeq 6000 sequencer (Illumina, Inc). Around 10,000 targeted cell recovery per sample has been applied to a single cell master mix with lysis buffer and reverse transcription reagents, following the Chromium NextGEM Single Cell 3' Reagent Kits User Guide, CG000204 Rev D (10X Genomics, Inc). Along with the single cell gel beads and partitioning oil, the single cell master mixture containing the single cell suspension was dispensed onto a Single Cell Chip G in separate wells, and the chip loaded to the Chromium Controller for GEM generation and barcoding, followed by cDNA synthesis and library preparation. At each step, the quality of cDNA and library has been examined by Bioanalyzer and Qubit. The resulting library has been sequenced in a custom program for 28b plus 91b paired-end sequencing on Illumina NovaSeq 6000.