Comparative framework and adaptation of ACME HS approach to single cell isolation from fresh-frozen endocrine tissue [dataset 2]

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 Salt (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 ACME HS followed by cryopreservation in 3xSSC*10% DMSO and methanol cell fixation. Key adaptations of the ACME HS method were the supplement of solution composition with 0.1M NAC and the introduction of additional washing steps in cold high salt 3xSSC* buffer. ACME dissociation was conducted in ~ 1 hours on a rotator at room temperature, with periodic pipetting of the solution. Afterward, we removed the ACME solution and washed the pellet with a two-step washing in cold 3xSSC. 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 platform, using the S4 Reagent Kit v1.5 and Illumina HiSeq2500 platform.