title: GSM6045583: sorted cells from LNs, CD, Hom samples, scRNAseq; Mus musculus; RNA-Seq
library_construction_protocol: For the scRNA-seq experiment of pooled lymph nodes (including mesenteric, paraaortic, inguinal, axillary, brachial and mandibular LNs), eight male Apoe-/-Ccl17wt/e and six Apoe-/-Ccl17e/e mice were fed on normal chow diet. Perfusion was performed with 5 mL PBS. Pooled lymph nodes from either  Apoe-/-Ccl17wt/e or Apoe-/-Ccl17e/e mice were mechanically crushed and passed through a 30 μm cell strainer using Hank's Medium (HBSS + 0.04% BSA) to obtain single cell suspensions, followed by B cell depletion using CD19 MicroBead. B cell-depleted fractions were stained with Viability Dye, anti-CD45, anti-CD3, anti-CD19 and anti-CD11c at 4°C for 20 minutes. Cells were washed and resuspended in Hank's Medium and then isolated by fluorescence-activated cell sorting, by gating for Live CD45+CD11c+CD3-CD19- cells. The cell suspension with viability > 80% was ready for subsequent single cell capture and library preparation. For the scRNA-seq experiment of para-aortic LNs, seven male Apoe-/-Ccl17wt/e and ten Apoe-/-Ccl17e/e mice were fed on 6-week HFD. Paraaortic LNs of same genotype were pooled and strained as aforementioned. Cells were resuspended in Hank's Medium, stained, and isolated by fluorescence-activated cell sorting, by gating for Live CD45+CD19-MHCII+ cells. The cell suspension with viability > 80% was ready for subsequent single cell capture and library preparation. Library was performed according to the manufacter's instructions (single cell 3' v3.1 protocol, 10x Genomics). Briefly, FACS-sorted cells  were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.