Marina Utkina ^1* Anastasia Shcherbakova ¹ Ruslan Deviatiiarov ¹ Alina Ryabova ¹ Marina Loguinova ¹ Valentin Trofimov ¹ Anna Kuznetsova ¹ Mikhail Petropavlovskiy ¹ Rustam Salimkhanov ¹ Denis Maksimov ² Eugene Albert ² Alexandra Golubeva ¹ Walaa Asaad ¹ Lilia Urusova ¹ Ekaterina Bondarenko ¹ Anastasia Lapshina ¹ Alexandra Shutova ¹ Dmitry Beltsevich ¹ Oleg Gusev ¹ Larisa Dzeranova ¹ Galina Melnichenko ¹ Ildar Minniakhmetov ¹ Ivan Dedov ¹ Natalia Mokrysheva ¹ Sergey Popov ¹

• ¹ Endocrinology Research Center, Moscow, Russia
• ² Lomonosov Moscow State University, Moscow, Moscow, Russia

Current dissociation methods for solid tissues in scRNA-seq studies do not guarantee intact single-cell isolation, especially for sensitive and complex human endocrine tissues. Most studies rely on enzymatic dissociation of fresh samples or nuclei isolation from frozen samples. Dissociating whole intact cells from fresh-frozen samples, commonly collected by biobanks, remains a challenge.Here, we utilized the acetic-methanol dissociation approach (ACME) to capture transcriptional profiles of individual cells from fresh-frozen tissue samples. This method combines acetic acid-based dissociation and methanol-based fixation. In our study, we optimized this approach for human endocrine tissue samples for the first time. We incorporated a high-salt washing buffer instead of the standard PBS to stabilize RNA and prevent RNase reactivation during rehydration. We named our approach ACME HS (ACetic acid-MEthanol High Salt). This technique aims to preserve cell morphology and RNA integrity, minimizing transcriptome changes and providing a more accurate representation of mature mRNA.We compared the ability of enzymatic, ACME HS, and nuclei isolation methods to preserve major cell types, gene expression, and standard quality parameters across 41 tissue samples. Our results demonstrated that ACME HS effectively dissociates and fixes cells, preserving cell morphology and high RNA integrity. This makes ACME HS a valuable alternative for scRNA-seq protocols involving challenging tissues where obtaining a live cell suspension is difficult or disruptive.