GSM8879392: 6_hr_control_rep1; Homo sapiens; RNA-Seq
Following treatment, cells were dissociated into a single cell suspension, cell counts and viability were determined with a fluorometric Nexcelom Cellometer automated cell counter (Nexcelom Biosciences, Lawrence, MA) using Acridine Orange (AO)/Propidium Iodide (PI) stain (Perkin Elmer, Inc., Waltham, MA), following manufacturer's instructions, and then the cells were processed for single-cell library construction. Cells were initially washed once in 37°C TrypLE (Thermo Fisher Scientific), aspirated, then incubated for ~20-25 min at 37°C in 0.25% Trypsin-EDTA (Thermo Fisher Scientific) with 1 mM DTT, 3 µg/ml actinomycin D, 25 µg/ml anisomycin, and 64 U/ml RNaseOUT recombinant ribonuclease inhibitor (Thermo Fisher Scientific, Waltham, MA) in 500 µl volume, gently agitating after 10 min of incubation. Using a wide-bore pipette tip, cells were then dissociated by gentle pipetting until no cell clumps were visible. After transferring to a 15 ml conical tube, 2 ml of 37°C base media was added to stop enzymatic activity, gently mixed by pipetting, and cells were pelleted at 200 x g for 3 min at 4°C. After media removal, cells were resuspended in 1 ml ice-cold cell suspension buffer (1X PBS, 0.5% Opti-Prep Density Gradient Medium (Sigma-Aldrich), 0.1% Bovine Serum Albumin Fraction V (Thermo Fisher Scientific, Waltham, MA), 64 U/ml RNaseOUT, 1mM DTT). The cells were pelleted twice more at 200 xg for 3 min at 4°C, washing each time with fresh Cell Suspension Media. After the final wash, cells were filtered through a 40 µm nylon cell strainer, transferred, and stored in a Lo-Bind tube.  Following dissociation, cell were counted and viability were determined with a fluorometric Nexcelom Cellometer automated cell counter. Single-cell HepaRG suspensions were barcoded and prepped for sequencing using the Chromium Next GEM Single Cell 3' Reagent Kit A (v3.1; 10X Genomics), following the manufacturer's instructions, with an input of 10,000 cells per sample. Then nanoliter scaled Gel Beads-in-emulsion (GEMs) were partitioned to capture single cells as well as a master mix of Illumina TruSeq Read 1 primers, 10X barcodes, unique molecule identifiers (UMI), poly(dT) primers, and reverse transcription enzymes, followed by cells lysing and a cDNA synthesis reaction. After contents were released from the GEMs, pooled barcoded cDNA was purified by silane magnetic beads and amplified by PCR. After fragmentation, Illumina Read 2, P5, P7, and a sample index were added to the pooled amplified cDNA before paired-end sequencing on an Illumina NovaSeq 6000, using a S2 flow cell. Sample library prep traces were verified and quantified on a Bioanalyzer High Sensitivity DNA chip (Agilent).