title: GSM8652520: EL pig endometrium1; Sus scrofa; RNA-Seq
library_construction_protocol: About 200 mg of endometrial tissue was clipped, washed twice with precooled phosphate-buffered saline (PBS) solution, placed in tissue protection solution (BioYou, 21903-10), and transferred to the laboratory on ice for follow-up treatment. After adding enzyme digestion solution (2 mg/mL collagenase II, 1 mg/mL dispersed protease, 30 μg/mL DNase I, 2.5 mM CaCl2, containing FBS 2% dissolved in basal medium), the samples were shaken in a 37 ℃ water bath shaker at 100 rpm/min for 20 min. The enzyme-digested suspension was filtered through a 30 µm cell filter and added to 15 mL PBS, centrifuged at 500 g for 5 min at 4 ℃. The supernatant was removed and the pellet resuspended with 1 ml of 0.04% BSA, added into 3 ml of 1X RBC to lyse erythrocytes, and left at room temperature for 5 min (the time can be adjusted according to the actual situation of erythrocyte lysis). The suspension was then washed twice with PBS and centrifuged at 4 ℃, 300 g for 5 min. The supernatant was removed and the pellet resuspended in 500 μl of 0.04% BSA. Cell parameters were then tested with a cell counter (CountessTM II FL Automated Cell Counter, Thermo Fisher Scientific, Waltham, MA, USA). The 10X Genomics 3 'transcriptome uses short-read long sequencing and microfluidic techniques to enable simultaneous transcriptomic profiling of 500-10,000 cells per sample. We combine Gel Beads containing barcode information with a mixture of cells and enzymes, which are then coated with oil surfactant droplets located in a microfluidic "double cross" system to form GEMs (Gel Beads-InEmulsions). GEMs flow into a reservoir and are collected, the gel beads dissolve to release the Barcode sequence, reverse transcrib the cDNA fragment, and label the sample. The gel beads were broken and the oil droplets were broken, and PCR amplification was performed using cDNA as template. The standard sequencing library was constructed by mixing all GEMs products