GSM8184976: MedullaryTumor_nuclei_1; Homo sapiens; RNA-Seq
Nuclei were isolated from fresh-frozen adrenocortical tumor, adrenal medullary tumor, and PitNET specimens. Fresh-frozen tissue samples were thoroughly minced on ice and placed into a gentleMACS C tube (Miltenyi Biotec) with 2ml ice-cold Hypotonic lysis buffer (10mM HEPES pH7.2, 5mM MgCl2, 10mM NaCl, 1% NP40). GentleMACS C tubes were then placed on the gentleMACS Dissociator (Miltenyi Biotec) and samples were homogenized by running the program h_mito_01, and then incubated on ice for 10 minutes. After repeating the homogenization step, 2 ml of Isotonic buffer (10mM HEPES pH7.2, 5mM MgCl2,10mM NaCl, 500mM sucrose) was added to the lysates, mixed by pipetting, filtered through a pre-wetted 70μm cell culture filter (Miltenyi Biotec) with Isotonic wash buffer (10mM HEPES pH7.2, 5mM MgCl2,10mM NaCl, 250mM sucrose) and centrifuged for 5 minutes at 1000g (4 °C). Then, we carefully removed the supernatant, resuspended the pellet in 1 ml DPBS with 1% BSA, and filtered it through a pre-wetted 30μm cell culture filter (Miltenyi Biotec). After all, the pellet was resuspended in DPBS containing 1% BSA volume 100-400 ul, depending on the pellet size. 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.