title: GSM8482693: GEX_Flot2WT naïve CD4+ T cells, stimulated (0, 0.25, 1 μg/ml), 3 hour; Mus musculus; RNA-Seq
library_construction_protocol: Cells stimulated with 0, 0.25, or 1 μg/ml of plate-bound anti-CD3 were hash-tagged with barcode sequence 'ACCCACCAGTAAGAC', 'GGTCGAGAGCATTCA', or 'CTTGCCGCATGTCAT', respectively, within each genotype (WT and Cre). The labeled cells were then pooled to create pooled sample WT and pooled sample Cre. For pooled sample WT and sample Cre, about 16000 live cells at 3x10^5 cells/ml concentration with more than 80% viabilities loaded into the Single Cell Chip followed by forming single cell emulsion in Chromium Controller (10x Genomics, 120263). The cDNA for antibody derived transcripts (ADT) and gene derived transcripts was generated and amplified according to manufacturer's instructions (10x Genomics and BioLegend). The Gene Expression library and the ADT Library were prepared using the Chromium single cell 3′ library and gel bead kit v3.1 (10x Genomics, 1000268) and additional reagents recommended in the protocol of Total A-seq (BioLegend). The two libraries were mixed at 9:1 molar ratio (Gene Expression library to ADT library) and sequenced by the NIEHS Epigenomics and DNA Sequencing Core Laboratory on NovaSeq 6000 (Illumina) with paired-end sequencing (Read 1: 28; Read 2: 90).