GSM9353086: DC-enriched afferent lymph, pre-BCG, Calf1; Bos taurus; RNA-Seq
Afferent lymph collected from the two timepoints, pre-BCG and post-BCG, was cryopreserved in foetal bovine serum with 10% dimethyl sulfoxide (DMSO) at -155 °C prior to cell sorting. Cells from afferent lymph were thawed and washed twice with PBS and then centrifuged at 1,200 RPM for 5 minutes. Cells were resuspended and stained with Zombie aqua (Biolegend) to assess cell viability. After being incubated with Zombie aqua for 15 minutes in the dark at room temperature, the washing step was repeated. Cells were resuspended with 1 mL of RPMI medium then placed through a cell strainer (70 µm, Falcon™) and placed on ice. Dendritic cells were enriched using Fluorescence-Activated Cell Sorting (FACS) to sort cells based on viability, high FSc and high side SSc properties, indicative of large cell size and high granularity, consistent with DC characteristics. Out of the total afferent lymph cell populations in each sample, approximately 14.02% were sorted as enriched DCs. A total of 1 x 105 sorted cells were utilised for scRNA-seq library preparation per sample, performed at the Queen's Medical Research Institute (QMRI). Approximately, 20,000 cells were loaded aiming for ~10,000 recovered cells per sample. Libraries were generated according to the 10x Genomics Single Cell v3 Reagent Kit protocol with 12 PCR cycles, followed by cDNA concentration measurement by Qubit. Each library was tagged with individual i7 indexes and underwent quality control using a Bioanalyser High Sensitivity Chip. Sequencing was performed on an S2 flow cell on the Illumina NovaSeq 6000 sequencing platform at Edinburgh Genomics.