title: GSM7634417: ScRNA-seq, mRNA library, Thymus; Mus musculus; RNA-Seq
library_construction_protocol: Single cell suspensions of thymus were obtained by scratching organs through a 70-μm nylon mesh cell strainer with a plastic plunge in PBS 5%BSA 1mM EDTA. Thymic cell suspensions were then incubated with red blood cell lysis buffer for 3 minutes at room temperature and enriched for CD4+ thymocytes by depletion of CD8+ and CD11c+ cells using the AutoMACS® Pro Separator with the Deplete program (Miltenyi). We used cell hashing with hashtag oligonucleotides (HTO) to multiplex the two samples from 2 individual mice. Cell surface staining used for gating cells from the Treg lineage and staining for distinct barcoded anti-mouse CD45 antibody (Biolegend; A0304 and A0305 for Foxp3eGFP and Foxp3eGFPxLta-/- Treg cells, respectively) were performed in PBS 5%BSA 1mM EDTA for 30 min on ice. For each sample, cells from the Treg lineage Live Dead-CD4+CD8-CCR6-CD3e+ expressing either CD25, Foxp3eGFP or both were bulk-sorted with BD FACS Aria II. Sorted cell samples (20,000 Foxp3eGFP Treg cells and 20,000 Foxp3eGFPxLta-/- Treg cells) were pooled with a target of 10,000 captured cells and loaded in a single capture well for subsequent 10x Genomics Single Cell 3' v3.1 workflow. Library was performed according to the manufacter's instructions (single cell 3' v3.1 protocol, 10x Genomics). Briefly, cells were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Illumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the appropriated sized fragments were selected with SPRIselect reagent. The pellet and supernatant fractions were separated for subsequent HTO and gene expression library construction. During the library construction, Illumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added. The HTO library was constructed using Truseq D701 and D702 sequences containing i7 indexes. The resulting libraries were pooled and sequenced on an Illumina NextSeq2000 platform with a P2 flow cell (100 cycles).