GSM3669249: Human NKT cell stimulated sample 3; Homo sapiens; RNA-Seq
Peripheral blood mononuclear cells (PBMCs) were isolated from fresh blood within 2 hours of collection using ficoll-Paque density gradient centrifugation. Single cell suspensions were stained with PE-hCD1d PBS-57 (NIH Tetramer Core Facility), followed by anti-PE microbeads (Miltenyi Biotec), and cell separation by autoMACS (Miltenyi Biotec). Separated NKT-enriched PBMCs were then stained with anti-hCD3 and hCD1d PBS-57 antibodies and NKT cells (CD3+CD1d PBS57+) were then sorted on BD FACSAriaII.  For stimulated NKT cells, freshly isolated PBMCs were first stimulated with PMA/Ionomycin for 2 hours, followed by NKT cell enrichment and sorting as aforementioned. Single cell sequencing libraries were generated using the 10X Genomics Chromium Single Cell 3' Reagent Kit (v2 Chemistry) and Chromium Single Cell Controller. Reverse transcription of the GEM (GEM-RT) was performed in a Thermocycler (Veriti™ 96-Well Fast Thermal Cycler, Applied Biosynthesis; 53 °C 45 min, 85 °C 5 min, 4 °C hold). cDNA amplification was performed after GEM-RT cleanup with Dynabeads MyOne Silane (Thermo Fisher Scientific) with the same Thermocycler (98 °C 3 min; 98 °C 15 sec, 67 °C 20 sec, 72 °C 1 min, repeat 12 cycles; 72 °C 1 min, 4 °C hold). Amplified cDNA is cleaned up with SPRIselect Reagent Kit (Beckman Coulter) and followed by library construction procedure, including fragmentation, end repaired, adaptor ligation, and library amplification. A Bioanalyzer (Agilent) was used for library quality control. Libraries were sequenced on an Illumina HiSeq4000 using paired-end flow cell: Read 1, 26 cycles, i7 index 8 cycles; Read 2: 110 cycles.