title: GSM8901257: PBMC, library GD009_1, GEX; Homo sapiens; RNA-Seq
library_construction_protocol: After overnight resting PBMCs were stimulated with extracts (10 µg/ml) or HDMPP (10 µg/ml), or medium alone as negative control, and incubated for 24h at 37ºC. After the incubation, cells were surface stained with fluorochrome-conjugated antibodies, for 40 minutes at 4°C using: anti-CD3 (AF700) (UCHT1), anti-CD4 (APCef780) (RPA-T4), anti-CD8 (PerCP-Cy5.5) (RPA-T8), anti-CD14 (V500) (M5E2), anti-CD19 (V500) (HIB19), anti-TCRγδ (PE-DA594) (IP26), anti-TCRδ1 (FITC) (TS8.2), anti-TCRδ2 (BV421) (B6), anti-CD137 (APC) (4B4-1), and anti-CD69 (BV605) (FN50) antibodies. TotalSeq-C oligonucleotide-conjugated antibodies (BioLegend) were also added at this step at 0.01mg/mL final concentration (one distinct antibody per sample). After two washes in PBS, cells were resuspended into 500 μl of MACS buffer (PBS containing 2mM EDTA (pH 8.0) and 0.5% BSA) and stored at 4ºC until flow cytometry acquisition. AIM+ (CD69+CD137+) Vδ1 and Vδ2 cells were captured by fluorescence-activated cell sorting (FACS), using a BD FACSAria Fusion cell sorter (Becton Dickinson). Samples were processed using 10x Genomics 5' end mRNA capture reagents (5v2.0 single cell gene expression profiling chemistry) as per manufacturer's recommendations; after droplet generation, and in-droplet based reverse transcription, cDNA was amplified by PCR for 13 cycles and gene expression library preparation followed. After quantification, equal molar concentration of each library was pooled and sequenced using the NovaSeq6000 Illumina sequencing platform to obtain 28- and 100-bp paired-end reads using the following read length: read 1, 100 cycles; read 2, 100 cycles; i7 index, 10 cycles and i5 index 10 cycles. Each gene expression library was sequenced aiming at a minimum mean sequencing depth of 30,000 reads per cell. Separate libraries were prepared to identify the DNA-Barcode that will allow de-multiplexing and linking each cell to a donor. Briefly, amplified DNA generated from antibody-DNA oligos was separated from transcriptomic cDNA based on size-selection following amplification. Antibody-DNA amplified fragments were less than 300 bp. Library preparation was followed in accordance with the manufacturer's recommendations. Each library was sequenced aiming for 5,000 reads per cell.
identifiers.namespace: GEO
identifiers.id: GSM8901257_r1