GSM8150730: minus, scRNAseq; Homo sapiens; RNA-Seq
LNs were collected from kidney transplantation recipients during living donor kidney transplantation. We have noticed that HLA-DR+ stromal cells represent only a small population within the stromal compartment of the lymph node. To ensure that sufficient HLA-DR+ stromal cells were included in the sorted cells used for sequencing, we included HLA-DR in the gating strategy of the sorting. We first gated on the HLA-DR+ (plus) and HLA-DR- (minus) stromal cells, subsequently we sorted DN, FRC, BEC and LEC based on PDPN and CD31 expression, resulting in two sorted cell suspensions. To assess the presence of a possible batch effect between these two samples, we added spike-in B cells in each sample. Library was performed according to the manufacter's instructions (single cell 3' v2 protocol, 10X Genomics). Briefly LNSCs 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 retrotranscribed to cDNA, whcih contains an Illumoiina R1 primer sequenc, Unique Molecular Identifier (UMI) and the 10X barcode. The pooled barcoded cDNA was then cleaned up with Dyna beads, amplified by PCR and the apropriate sizxed fragment were selected with SPRI select reagent for subsequent library construction. During the library construction Illumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and sample index were added. Single-cell RNA-sequencing libraries were prepared according to the manufacturer's instructions (CG000204 Rev D) using Chromium Next GEM Single Cell 3ʹ GEM, Library & Gel Bead Kit v3.1 (10X Genomics, PN-1000121) and Chromium Next GEM Chip G Single Cell Kit (10X Genomics, PN-1000120)