GSM4908723: Mouse liver iNKT cell sample 2; Mus musculus; RNA-Seq
Murine peripheral organs, including spleen, liver and lymph nodes, were harvested from 5 weeks old C57BL/6 mice. For the enrichment of iNKT cells, total cells were stained with biotin-conjugated anti-mouse CD8 Ab, anti-mouse B220 Ab and anti-biotin magnetic beads (eBioscience). Negatively selected CD8- and B220- cells were then stained with anti-mouse TCR-b, CD1d-Tetramer Abs. iNKT cells of whole population were further sorted from C57BL/6 mouse peripheral organs using FACSAria II Usage, cells collected with purity greater than 97%. 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. Single-cell RNA-Seq