GSM8973635: MCD, replicate 4, 5prime, cohort 2; Homo sapiens; RNA-Seq
Lymph node resections with clinicopathological diagnoses of UCD, MCD, and non-malignant non-infectious reactive lymph nodes (RLN) were identified from the institutional archives. We identified cases where rapid intraoperative frozen section diagnosis was conducted on whole lymph node resections. Upon removal from the patient, lymph node sections were embedded in Optimal Cutting Temperature (OCT) compound and frozen within a 10-minute window per clinical intraoperative diagnostic requirements that also ensured high-quality RNA reflecting the in-vivo state. The lymph node was bisected along its long axis. One-half of the bisected lymph node was frozen, while the other half was fixed in formalin and subsequently embedded in paraffin (FFPE). The OCT-frozen half was used for single-nuclei transcriptomic analysis, while the corresponding FFPE half was used for single-cell proteomic imaging (Supplemental Figure 1). Frozen tissue was used for DNA sequencing and SNP array analysis Single nuclei RNA was processed for sequencing by constructing gene expression (GEX) libraries (Chromium Next GEM Single Cell 3ʹ Kit v3.1, PN-1000268) or gene expression and V(D)J immune profiling libraries (Chromium Next GEM Single Cell 5' Kit v2, PN-1000263; Chromium Single Cell Human TCR Amplification Kit , PN-1000252; Chromium Single Cell Human BCR Amplification Kit, PN-1000253). Briefly, the single nuclei suspension was mixed with RT Master Mix and loaded with barcoded single-cell 3' or 5′ gel beads and partitioning oil onto microfluidic chips to encapsulate 5000 nuclei per sample using Chromium Controller. After reverse transcription and cleanup, cDNA libraries were generated according to manufacturer instructions with one additional cycle of PCR amplification to account for the relatively lower amount of RNA in nuclei than in whole cells. cDNA was fragmented and end-repaired, size-selected and PCR amplified to generate a 3' or 5′ gene expression library. For VDJ library construction, full-length TCR or BCR transcripts were enriched from 4μL of amplified cDNA, and 50 ng of enrichment product was used for library construction.