title: GSM3733525: NKT_mm_Thymus_St2_2: Mouse thymus stage 2 iNKT cell sample 2; Mus musculus; RNA-Seq
library_construction_protocol: Murine thymi were harvested from 5 weeks old C57BL/6 mice or Vα14 transgenic mice. The thymus iNKT cells were enriched by positive selection using biotinylated antibodies against PE. Stage 1(CD24-CD44-NK1.1-), stage 2(CD24-CD44+NK1.1-) and stage 3(CD24-CD44+NK1.1+) were sorted  from 5 weeks C57BL/6 mouse thymus, and Stage 0 iNKT cells (CD24+NK1.1-) were sorted from 5 weeks old Vα14 transgenic mouse thymus through BD FACS AriaII. 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.
attributes.tag: GEO Accession
attributes.value: GSM3733525