GSM8256999: mRNA-LNP, HTO; Mus musculus; RNA-Seq
Single cell suspensions were incubated with 1 μg ml−1 anti-CD16/32 in PBS supplemented with 0.5% BSA and 2 mM EDTA (PBE) for 5 min at room temperature and then stained for cell surface markers at 4 °C for 20 min in PBS. For single-cell transcriptomic analysis, stained cells were further incubated with DNA-barcoded anti-biotin and sample hashtag (anti-MHC-I) antibodies (BioLegend) for 20 minutes in PBE, washed three times with PBE, and bulk-sorted. Sorted DCs were collected into a microfuge tube with 300 μl PBS supplemented with 0.4% BSA. After the sort, tubes were topped with PBS 0.4% BSA, centrifuged and the buffer was carefully reduced by removing the volume with a pipette to a final volume of 40 μl. Cells were counted for viability and immediately submitted to library preparation. The scRNA-seq library was prepared using the 10X Genomics Single Cell Chromium 5' system, according to the manufacturer's instructions, at the Genomics Core of Rockefeller University and was sequenced on an Illumina NovaSeq SP flowcell to a minimum sequencing depth of 30,000 reads per cell using read lengths of 26 bp read 1, 8 bp i7 index, 98 bp read 2.