GSM7910662: B. burgdorferi Infected; Mus musculus; RNA-Seq
Single-cell suspension of lymphocytes was produced by mechanically homogenising the pooled popliteal lymph nodes with a custom-made metal cell strainer to detach cells from the stroma. Cells were washed twice with PBS with 2 % fetal calf serum (FCS) buffer and filtered through 77 µm silk. Cells were re-filtered through a 40 µm filter and suspended in RPMI medium with 2 % FCS buffer. Extracted lymphocytes were processed with the Chromium platform (10X Genomics) for library preparation, and the barcoded scRNA-seq libraries were constructed using 10XGenomics Chromium Next GEM Single Cell 3', Reagent Kit v3.1. Briefly, reverse transcription was performed to specific single-cell gel beads in the emulsion to produce full-length 10X barcoded cDNA from polyadenylated mRNA. The cDNA was amplified with PCR, followed by enzymatic fragmentation, end repair and A-tailing, adaptor ligation, and sample index PCR. Qualities of cDNA were ensured with Agilent Bioanalyzer 2100, and index PCR was made using Chromium i7 Multiplex Kit. Prepared libraries were sequenced using the Illumina Novaseq6000 Sequencing System