GSM8516539: pituitary, snRNAseq; Panthera tigris altaica; RNA-Seq
The thawed pituitary tissue was carefully transferred to a new 50 mL conical tube containing Hibernate E®/B27®/GlutaMAX™ (HEB) medium until the tissue settled to the bottom of the tube. The HEB medium was then decanted into a new 50 mL conical tube, retaining only sufficient medium to cover the tissue. The retained HEB medium was reserved for subsequent experiments. The tissue was treated with 10 mL of trypsin solution and incubated at 37ºC for 20 minutes. Following incubation, the trypsin solution was removed and discarded. At this stage, the previously reserved HEB medium was added to the tissue, which was then pipetted into a silanized Pasteur pipette containing the HEB medium, and the contents were immediately returned to the conical tube. The tissue was passed through the pipette 10-15 times, after which it was allowed to settle for 1 minute. The supernatant, containing the dispersed cells, was transferred to a new conical tube, leaving the tissue fragments at the bottom. The cells were then centrifuged at 200 rcf for 2 minutes, and the supernatant was removed and discarded. The resulting cell pellet was resuspended in pre-warmed 10% FBS/DMEM medium. For nuclear isolation from the single-cell suspension, cells were centrifuged again at 300 rcf for 5 minutes. The supernatant was discarded, and Lysis Buffer was added until the cells were fully suspended. Cell lysis was continued on ice for 5 minutes before centrifuging the nuclei at 500 rcf for 5 minutes at 4°C. The supernatant was subsequently removed, and Cell Nucleus Wash and Resuspension Buffer was added, with gentle pipetting and mixing. The nuclei were then centrifuged at 500 rcf at 4°C for 5 minutes. Following this, the supernatant was removed and Nucleus Wash and Resuspension Buffer, or an appropriate volume of buffer, was added to the precipitated nuclei to achieve the target concentration of 1 nucleus/µL. Cell debris and large cellular fragments were removed using a cell filter. Nuclei concentration was determined using a Countess II FL automated cell counter, achieving a pituitary nuclei concentration of 1080 nuclei/µL. The nuclei were kept on ice, and the 10x Genomics® Single Cell protocol was performed immediately to minimize the interval between nucleus preparation and chip loading. Library for sequencing was prepared using the 10x Genomics Chromium system with Chromium Next GEM Single Cell 3' Reagent Kits v3.1 (CG000204, 10x Genomics) and follow the User Guidelines. In summary, the library construction process was conducted in three main steps: GEM Generation & Barcoding, Post GEM-RT Cleanup & cDNA Amplification, and 3ʹ Gene Expression Library Construction. Finally, the total cDNA quantity was determined by analyzing 1 µl of the sample on an Agilent Bioanalyzer using a High Sensitivity microarray chip, following dilution of the sample with nuclease-free water at a 1:10 ratio. The expression libraries were constructed with standard Illumina paired-end structures, ultimately yielding a standard Illumina BCL data output folder.