title: GSM8617646: Smartseq2_632; Mus musculus; RNA-Seq
library_construction_protocol: Lymph nodes and gut tissue were dissected, finely chopped, and digested in RPMI medium with Collagenase D to obtain single-cell suspensions. For gut-associated lymph nodes, duodenal lymph nodes were identified using anatomical markers. Dendritic cells were isolated from the duodenal lamina propria by separating intestines, removing mucus with PBS and DTT, and stripping epithelium with EDTA. The tissue was further digested with DNaseI and collagenase, followed by Percoll gradient centrifugation to isolate DCs. Single-cell sorting of DCs was conducted into 96-well plates with TCL buffer and stored at –80°C. Library Preparation for Single-cell RNA-seq (SmartSeq2). Single cells were sorted onto plates, covering all conditions in each replicate. RNA was extracted using RNAClean XP SPRI beads, then reverse-transcribed into cDNA with RT and TSO primers. The cDNA was amplified, cleaned with SPRI beads, and tagmented using the Nextera XT DNA Library Preparation Kit (Illumina). Barcoded libraries were pooled and sequenced on the Illumina Nextseq 550 platform. Library Preparation for Single-cell RNA-Seq (10x Genomics). Cells were labeled with hashtag oligonucleotide (HTO)-tagged antibodies, sorted, and counted for viability. scRNA-seq libraries were generated using the 10x Genomics Chromium system, following the manufacturer's protocol. Libraries were sequenced on an Illumina NovaSeq SP flowcell at a minimum depth of 30,000 reads per cell. Library Preparation for Bulk RNA-seq. RNA was isolated using RNAClean XP beads. Reverse transcription was performed with specific primers, followed by cDNA amplification with KAPA HiFi HS Ready Mix. The amplified product was cleaned with RNAClean XP beads and tagmented using the Nextera XT DNA Library Preparation Kit (Illumina). Libraries were pooled and sequenced as 75-base single-end reads on an Illumina NextSeq platform.