title: GSM9055915: adrenal gland 1, 10x; Mus musculus; RNA-Seq
library_construction_protocol: A patient's whole blood sample was diluted in PBS at a ratio of 1:1. Ficoll solution was added to a clean 15 ml falcon. The diluted blood sample was carefully layered on the Ficoll layer, preventing their mixing. Then, the sample was centrifuged with the following parameters: 450 g, 30 minutes, without brake. After that, the mononuclear cell ring was carefully collected from the Ficoll surface with a dispenser, and transferred to a clean 15 ml tube. The sample volume was brought to 10 ml using PBS. The sample was centrifuged at the parameters: 300 g for 10 minutes, after which the supernatant was removed. These steps were performed twice. Cryopreservation of the isolated cells was carried out according to the manufacturer's protocol. The samples were defrosted, washed, samples from 4 patients were pooled with 1 million cells from each patient for further loading onto the chip. Murine adrenal glands were excised and placed in PBS 1X with 10% fetal bovine serum (FBS) on ice. Under a binocular microscope, adipose tissue was removed with two tweezers/scalpel, and adrenal glands were mechanically homogenized with a scalpel on ice. Next, the adrenal gland homogenates were placed in a dissociating solution (HBSS buffer, Ca2+ 1.26 mM, collagenase IV 20 mg/mL, DNAse I 0.7 mg/ul, 0.5% BSA, RNase inhibitor) followed by 30-minute incubation at 37°C in a shaker-incubator (100 RPM). During the incubation, the suspension was pipetted every 10 minutes. Following the incubation, it was carefully pipetted, and 800 μl of PBS 1X with 10% FBS were added. The suspension was centrifuged 450 rcf for 6 min at +4◦C, then the supernatant was decanted, and the pellet was resuspended in 700 μl PBS 1X with 10% bovine serum and passed through a 70 μm filter. Then it was centrifuged 450 rcf 6 min at +4◦C, the supernatant was collected, the pellet was resuspended in 450 μl 1x PBS-0.05% BSA. The cell suspensions were gently mixed with cooled methanol in a ratio of 1:10 and placed for long-term storage at −80°C. For further work, the samples were defrosted on ice and rehydrated using a citrate buffer. Human pancreatic neuroendocrine tumor tissue was dissociated for single cell RNA sequencing using the Multi Tissue Dissociation Kit 2 by Miltenyi Biotec (cat. no. 130-110-203). Briefly, the tissue was placed in a petri dish on ice and cut into small pieces of 2–4 mm. The pieces were infused with the RPMI/enzyme mix (Miltenyi Biotec), transferred to a gentleMACS C tube containing RPMI/enzyme mix, attached to the sleeve of the single cell suspension dissociator (DSC-400) and run using the standard “H_Tumor_heater_1” program. After the termination of the program, the cells were centrifuged at 650 x g for 6 min at +4°C, resuspended in PBS 1X with 0.04 % bovine serum, passed through a 70 µm strainer and centrifugation was repeated. The supernatant was collected, the pellet was resuspended in 1 ml 1x PBS-0.04% BSA. Next, the cell suspension was fixed in cooled methanol. The cell suspension obtained in the previous step was mixed with methanol in a ratio of 1:10 and placed for long-term storage at −80°C. For further work, the samples were defrosted on ice and rehydrated using a citrate buffer. The Chromium Next GEM Single Cell 5′ v2 Kit, Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 (10× Genomics, Pleasanton, CA, USA) and the Chromium Single Cell Controller Instrument were used to generate 10× single-cell libraries according to the manufacturer's instructions. The SeekOne DD Single Cell 3' Transcriptome-seq Kit v1.1 and the SeekOne Digital Droplet System Instrument were used to generate single-cell libraries according to the manufacturer's instructions. The MobiCube Single-Cell 3' RNA-seq Kit v2.0, MobiCube Single-Cell 5' V(D)J Kit v1.0 and the MobiNovaSP-100 Instrument were used to generate single-cell libraries according to the manufacturer's instructions.