NextSeq 500 paired end sequencing; Single-cell RNA sequencing data (SORT-seq) of Treg cells in the human postnatal thymus
Thymic tissue was finely dissociated with scissors at room temperature, placed in gentleMACS C tubes (Miltenyi Biotech), filled up to 10 mL with MACS buffer (PBS containing 0.5% FCS) and mechanically disrupted with the gentleMACS Dissociator (Miltenyi Biotech). A single-cell suspension was generated by pressing the tissue through a 100 μm cell strainer. Cells were collected in 50 mL Falcon tubes, pelleted through centrifugation (350g for 10 minutes at RT) using MACS buffer and counted. Single cells were FACS-sorted into 384-well plates containing 384 primers and Mineral oil (Sigma). After sorting, plates were snap-frozen on dry ice and stored at -80° C.  For amplification, cells were heat-lysed at 65° C followed by cDNA synthesis using the CEL-Seq2 protocol (Hashimshony et al. 2016) and robotic liquid handling platforms. After second strand cDNA synthesis, the barcoded material was pooled into libraries of 384 cells and amplified using IVT. Following amplification, the rest of the CEL-seq2 protocol was followed for preparation of the amplified cDNA library, using TruSeq small RNA primers (Illumina).