GSM1923497: Single_cell_RNA-seq_NKT1_8; Mus musculus; RNA-Seq
For RNA isolation from thymic iNKT cell subsets, thymus cell suspensions were enriched for iNKT cells by negative selection of cells expressing CD8b.2, CD19 or TER-119 via magnetic selection utilizing reagents and equipment from Stem Cell Technologies. The remaining cells were then stained using an 11-parameter panel of reagents that included tetramers of CD1d loaded with either aGalcer or PBS57 combined with streptavidin-brilliant violet (BV)421 (produced in-house or obtained from the NIH Tetramer Core Facility), anti-CD44-V500 (BD Biosciences #56780), live/dead yellow (ThermoFisher Scientific #L34959), anti-CD4-Qdot 605 (ThermoFisher Scientific #Q10092), anti-CD103-FITC (Affymetrix #11-1031), anti-CD24-PerCPCy5.5 (Affymetrix #45-0242), anti-CD27-APC (Affymetrix #E07114), anti-CD8a-AF700 (Biolegend 100730), anti-TCRb-APC-eF780 (Affymetrix #47-5961), anti-CCR6-PE (Biolegend #129803), and anti-NK1.1-PE-Cy7 (BD Biosciences #552878).  iNKT subsets were sorted using a FACSaria III (BD Biosciences). Single cell RNAseq was perfromed as described in Picelli et al.Nat Protoc. 2014, PMID:24385147. We performed 22 cycles of amplification. Single cell RNAseq was perfromed as described in Picelli et al.Nat Protoc. 2014, PMID:24385147. Barcoded Illumina sequencing libraries (Nextera XT library preparation kit, Illumina) Whole Transcriptome Amplification (SmartSeq2 - Picelli et al.2014); Libraries were sequenced on the HiSeq2500 Illumina platform to obtain 50-bp single end reads (TruSeq® Rapid Kit, Illumina).
GEO Accession
GSM1923497