GSM8826135: HTO_derived_cDNA; Mus musculus; OTHER
At D42, migDC (sorted as alive B220- CD11c+toint MHCIIhi) and CD4+ cells (sorted as alive B220-CD11c-MHCII-CD8a-CD4+) were enriched by FACS sorting from BP and BPT tdLN. BP CD4+ cells were labeled with TotalSeq™ barcoded antibody A0301 Hashtag1 (H1), BP migDC with A0302 Hashtag2 (H2), BPT CD4+ cells were labeled with A0303 Hashtag3 (H3) and BPT migDC with A0304 Hashtag4 (H4). The 4 populations were pooled at the ratio 1:1:1:1 (7500 cells per population) before being processed for multiplex scRNA-seq. For both librairies, cells were labelled with TotalSeq™-A Cell Hashing antibodies (BioLegend, Amsterdam, The Netherlands) as described. Labelled and washed cells were loaded onto the Chromium iX targeting 16000 captured cells per sample and processed using Single Cell 3' v3.1 (dual index) workflow according to the manufacturer's instructions up until the cDNA amplification step (10X Genomics, Leiden, The Netherlands). The following modifications were made in the next steps. Two picomoles of HTO (5'GTGACTGGAGTTCAGACGTGTGC*T*C) cDNA primers were added into the cDNA amplification PCR, and cDNA was amplified according to the 10x Genomics Single Cell 3' v3.1 protocol using 11 cycles. Following PCR, SPRIselect reagent (Beckman-Coulter, Villepinte, France) at a ratio of 0.6X was used to separate the cDNA fraction derived from cellular mRNAs (retained on beads) from the Cell Hashtag (HTO)-containing fraction (in supernatant). The cDNA fraction was processed according to the 10x Genomics Single Cell 3' v3.1 protocol to generate the transcriptome library. Additional SPRI beads reagent was added to the HTO fraction to bring the ratio up to 2.1X. The beads were washed with 80% ethanol. After final elution in EB buffer, separate PCR was carried out using the 2x KAPA HiFi PCR Master Mix (Roche Sequencing Solutions) to generate dual index cell hashing HTO library according to BioLegend's recommandations. Then surplus PCR primers were further removed by purification using SPRIselect beads. The final mRNA and HTO libraries were checked for quality and quantified using Bioanalyzer 2100 system (Agilent technologies, Les Ulis, France).