For FACS sorting of isolated thymus cells, dissociated cells were stained with a panel of antibodies prior to sorting based on CD45 or CD3 expression gate. Enrichment of EpCAM-positive cells was performed using CD326 (EpCAM) microbeads (Miltenyi Biotec, 130-061-101) according to manufacturer’s protocol. CD45 depleted cells were obtained using CD45 microbeads (Miltenyi Biotec, 130045-801) according to manufacturer’s protocol. Cell number and viability were checked after the enrichment to ensure that no significant cell death has been caused by the process.
For the droplet-encapsulation scRNA-seq experiments, 8000 live, single, CD45+ or CD45− FACS-isolated cells or MACS-enriched cells were loaded on to each of the Chromium Controller (10x Genomics). Single-cell cDNA synthesis, amplification, and sequencing libraries were generated using the Single Cell 3’ and 5’ Reagent Kit following the manufacturer’s instructions. The libraries from up to eight loaded channels were multiplexed together and sequenced on an Illumina HiSeq 4000. The libraries were distributed over eight lanes per flow cell and sequenced using the following parameters: Read1: 26 cycles, i7: 8 cycles, i5: 0 cycles; Read2: 98 cycles to generate 75-bp paired-end reads. For the plate-based scRNA-seq experiments, a slightly modified Smart-Seq2 protocol was used. After cDNA generation, libraries were prepared (384 cells per library) using the Illumina Nextera XT kit. Index v2 sets A, B, C, and D were used per library to barcode each cell for multiplexing. Each library was sequenced (384 cells) per lane at a sequencing depth of 1 to 2 million reads per cell on HiSeq 4000 using v4 SBS chemistry to create 75-bp paired-end reads.
For the droplet-encapsulation scRNA-seq experiments, 8000 live, single, CD45+ or CD45− FACS-isolated cells or MACS-enriched cells were loaded on to each of the Chromium Controller (10x Genomics). Single-cell cDNA synthesis, amplification, and sequencing libraries were generated using the Single Cell 3’ and 5’ Reagent Kit following the manufacturer’s instructions. The libraries from up to eight loaded channels were multiplexed together and sequenced on an Illumina HiSeq 4000. The libraries were distributed over eight lanes per flow cell and sequenced using the following parameters: Read1: 26 cycles, i7: 8 cycles, i5: 0 cycles; Read2: 98 cycles to generate 75-bp paired-end reads. For the plate-based scRNA-seq experiments, a slightly modified Smart-Seq2 protocol was used. After cDNA generation, libraries were prepared (384 cells per library) using the Illumina Nextera XT kit. Index v2 sets A, B, C, and D were used per library to barcode each cell for multiplexing. Each library was sequenced (384 cells) per lane at a sequencing depth of 1 to 2 million reads per cell on HiSeq 4000 using v4 SBS chemistry to create 75-bp paired-end reads.
