title: GSM5554950: Ctr_LTi_rep2; Mus musculus; RNA-Seq
library_construction_protocol: (WT/KO:PD-1-high/PD-1-neg): Single cell suspension were obtained from the colon lamina propria of C57BL/6 mice. For sorting huPD-1high LTi and huPD-1- LTi cells, cells were stained with relative antibodies followed by sorting on a BD Aria III cell sorter for "live Lineage-CD127+KLRG-1-c-Kit+CCR6+huPD-1high" and "live Lineage-CD127+KLRG-1-c-Kit+CCR6+huPD-1-" cells.(C-1, C-2, P-1, P-2): Single cell suspension were obtained from the small intestinal lamina propria of B6 mice. For sorting LTi cells, cells were stained with relative antibodies followed by sorting on a BD Aria III cell sorter for "live Lineage-CD127+KLRG-1-c-Kit+CCR6+"siLP-ILC: Single cell suspension were obtained from the small intestinal lamina propria of C57BL/6 mice. For sorting ILC, cells were stained with relative antibodies followed by sorting on a BD Aria III cell sorter for "live Lineage-CD127+“ Bulk RNA-Seq: The sorted LTi were collected in lysis buffer of the SMART-Seq HT Kit (Takara) followed with cDNA synthesis and amplification according to the manufacturer's protocols. Sequencing libraries were generated using NEB Next Ultra DNA Prep Kit (New England Biolabs), according to the manufacturer's instruction. Libraries were assessed using the Agilent 2100 Bioanalyzer system and sequenced by paired-end 150 base pair reads using the Illumina NovaSeq platform. single-cell RNA-Seq: Small intestine lamina propria lymphocytes were stained for ILC sorting (live lineage-CD127+) by FACSAria III Cell Sorter (BD Biosciences). The viability of sorted cells was assessed as >90%. Single cell capture and cDNA synthesis were performed on the BD Rhapsody Single-Cell Analysis System (BD), according to the manufacturer's instruction. Then, the single-cell library was generated and sent to sequencing on Illumina NovaSeq platform.