Comparative framework and adaptation of ACME HS approach to single cell isolation from fresh-frozen endocrine tissue.

Current dissociation methods for scRNA-seq studies of solid tissues do not guarantee intact single-cell isolation from fresh frozen samples, especially for sensitive and heterogeneous endocrine tissues. Here, we adapted the acetic-methanol dissociation method – ACME High Soult (ACME HS) to isolate intact single cells from fresh-frozen endocrine tumor samples. We compared enzymatic, ACME HS, and nuclear isolation methods ability to preserve major cell types and gene expression. We demonstrated that ACME HS dissociates and fixes cells, preserving cell morphology and high RNA integrity. This renders ACME HS a valuable alternative in the scRNA-seq protocols for challenging tissues. Single cells were isolated from tissue samples using enzymatic dissociation method. Approximately 5-250 mg of fresh tissue samples were washed in HBSS, thoroughly minced on ice, and placed in dissociating solution at 37°C with gentle pipetting every 5 minutes. Tissue samples were dissociated with 25-30 ul enzyme D Multi Tissue Dissociation Kit (MTDK) (Miltenyi Biotec) or enzyme A Neural Tissue Dissociation Kit P (NTDK) (Miltenyi Biotec) or 2 mg/ml collagenase IV (Gibco, Thermo Fisher Scientific) or collagenase I (Gibco, Thermo Fisher Scientific) in 870 mM HBSS, 10% FBS, 20 mM HEPES for 20-30 minutes. Obtained homogenate was filtered in 3-5ml of Wash Buffer (1x DPBS, containing 10% FBS, 20mM HEPES, 6mM glucose) through a pre-wetted 70μm cell culture filter (Miltenyi Biotec) and centrifuged for 5 minutes at 300xg (4 °C). After all, the pellet was resuspended in a Wash Buffer volume of 100-400 ul, depending on the pellet size. Single cells or nuclei were captured and barcoded, and cDNA libraries were generated using the Chromium Next GEM Single Cell 3ʹGEM, Library & Gel Bead Kit v3.1 (10X Genomics) according to the manufacturer’s instructions. Final libraries were multiplexed and sequenced on an Illumina Novaseq 6000 / Hiseq 2500 platform, using the S4 Reagent Kit v1.5.