Single cell library preparation and sequencing

Single Cell RNA-Seq was performed using the 10X Genomics Chromium Next Gem Single cell 3′ Reagents Kits v3.1 (PN-1000121, {"type":"entrez-nucleotide","attrs":{"text":"CG000315","term_id":"33868963","term_text":"CG000315"}}CG000315 protocol rev A, 10X Genomics) and Dual Index kit TT set A (PN- 1000215, 10X Genomics) according to the manufacturer’s instructions. For the resting and stimulated samples, the target was estimated at 8000 cells, while for the fresh and frozen samples, the target was set at 6,000 cells. Briefly, the cell suspension barcoded gel beads and partitioning oil were loaded onto the 10X Genomics Chromium Chip (Next GEM chip G) to generate single cell Gel Beads In emulsion (GEMs). Captured cells were lysed and the transcripts were barcoded through reverse transcription inside individual GEMs. The constructed libraries were sequenced on Illumina NextSeq500 (FC High output 300 cycles) or Novaseq (FC S1 standard 200 cycles) and processed using Cell Ranger version 6.0.1 (10X Genomics).