GSM6605131: adult female naked mole-rat thoracic thymus short-read 10x scRNA-seq, replicate 4; Heterocephalus glaber; RNA-Seq
Thoracic tissue was dissected using sterile surgical techniques and subsequently transferred to a petri dish containing PBS. Fat tissue surrounding the thymic parenchyma was carefully removed with microscissors and tissue forceps under a surgical microscope. Excess liquid/PBS was carefully removed with Kimwipes before weight measurement. For histological analysis, the tissues were fixed in 10% formalin and following standard tissue processing procedures. For the purpose of creating single-cell suspensions, thymic tissues were ground through 100 μm and 40 μm cell strainers (Falcon 352360 and 352340, Corning, NY) with a syringe plunger. Cells were subsequently pelleted (300g, 3 minutes, 4 ̊C), resuspended in 5 mL of ACK lysis buffer (Lonza BP10-548E, Basel, Switzerland) for 5 minutes at room temperature, and washed and resuspended with PBS with 0.04% BSA. Cell density and viability were determined using the Countness II Cell Counter (Thermofisher AMQAF1000). The cells were pelleted and resuspended at 1 million cells per ml of 0.04% BSA/PBS prior generating the 10× GEM single-cell libraries. Single cells were captured in droplet emulsion using the Chromium Controller (10× Genomics, Pleasanton, CA), and GEM single-cell libraries were constructed according to the 10× Genomics protocol using the Chromium Single-Cell 30 Gel Bead and Library 3' V3 kit (10× Genomics, Pleasanton, CA). In brief, cell suspensions were diluted in PBS with 0.04% BSA to a final concentration of 1×106 cells/mL (1,000 cells per μL). Cells were loaded in each channel with a target output of 10,000 cells per sample. All reactions were performed in a C1000 Touch Thermal Cycler (Bio-Rad Laboratories, Hercules, CA) with a 96 Deep Well Reaction Module. Twelve cycles were used for cDNA amplification and sample index PCR. Amplified cDNA and final libraries were evaluated using a Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA) with a high sensitivity chip. paired-end ilimina next-seq