Sequencing of murine corticotrophs from male POMC-GFP mice subjected to chronic restraint stress
Sequencing of murine corticotrophs from male POMC-GFP mice subjected to chronic restraint stress
Anterior pituitary cells were acutely isolated by trypsin digestion. Each cell preparation used anterior pituitary glands collected from three animals. After dissociation, cells were resuspended in 500 μL of phosphate-buffered saline supplemented with 25 mM HEPES and 5 mM EDTA (FACS buffer) and passed through a 35-μm cell strainer that was washed with a further 200 μL of the FACS buffer. Draq7 was added as a vitality marker and cells sorted using a SH800 Cell Sorter (Sony). Gates were established using wild-type pituitaries to avoid capturing eGFP negative (−ve) cells and to select single cells. Sorted single cells were resuspended into low bind Eppendorf tubes into 300 μL of Trizol and were frozen on dry ice and stored at −80 °C until sending for sequencing. RNA isolation was conducted at Azenta Life Science (South Plainfield, NJ). Total RNA was extracted using Qiagen RNeasy Plus Mini kit following manufacturer's instructions (Qiagen, Hilden, Germany). Extracted RNA samples were quantified using Qubit Fluorometer (Life Technologies, Carlsbad, CA, USA) and RNA integrity was checked using Agilent TapeStation (Agilent Technologies, Palo Alto, CA, USA). Library preparation was conducted at Azenta Life Science (South Plainfield, NJ). Ultra-low input RNA sequencing library was prepared by using SMART-Seq v4 Ultra Low Input Kit for Sequencing was used for full-length cDNA synthesis and amplification (Clontech, Mountain View, CA), and Illumina Nextera XT (Illumina, San Diego, CA, USA) library was used for sequencing library preparation. Briefly, cDNA was fragmented, and adaptor was added using Transposase, followed by limited-cycle PCR to enrich and add index to the cDNA fragments. The sequencing library was validated on the Agilent TapeStation (Agilent Technologies, Palo Alto, CA, USA), and quantified by using Qubit Fluorometer (ThermoFisher Scientific, Waltham, MA, USA) as well as by quantitative PCR (KAPA Biosystems, Wilmington, MA, USA).