GSM9150192: Single-cell samples indexed with RPI-45; Homo sapiens; RNA-Seq
Thymus tissues were harvested from consented MCHTB (Melbourne Children's Heart Tissue Bank) patients undergoing heart surgery according to approved protocols (HREC38192). Thymic cell suspensions were incubated with anti-mouse 6B11-PEcy7 (or 6B11-PE). PE or PE-cy7 positive cells were enriched using MACS enrichment, stained for flow cytometric sorting, sorted into individual thymic stages for low cell or single cell RNA-sequencing. Matched blood from thymic donors were cell sorted for single cell sequencing without prior MACS enrichment. Single cell transcriptome libraries were generated as described previously [Perriman et. al, 2023]. In brief, the samples were flow-sorted into a 384-well polymerase chain reaction (PCR) plate for complementary DNA (cDNA) synthesis. Samples were then pooled on a per plate basis for second-strand DNA synthesis and in-vitro transcription (IVT). The amplified RNA was fragmented, and during the following reverse transcription a 5'-tag sequence was introduced to facilitate the final library amplification. The libraries were sequenced on an Illumina NextSeq2000 sequencer with the following read parameters: read1:15bp, index read: 6bp, read2:67 bp.