Instrument: Illumina HiSeq X Ten
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: PAIRED
Construction protocol: We extracted the total RNA using the Trizol method. We added 1 ml of Trizol (invitrogen) to the sample (4-7 million cells) stored at -80 ° C, and carefully piped the sample with a pipette, which was the homogenate of the tissue cells. Then, 200 μl of chloroform was added to the homogenate, and the sample was shaken for 15 seconds using a vortex shaker until the phase separation disappeared. After standing at room temperature for 2-3 minutes, the sample was centrifuged at 12,000 g for 15 minutes at 4 °C. The colorless supernatant was transferred to another new centrifuge tube, mixed well with an equal volume of isopropanol, held at -20 ° C for 10 minutes, and then centrifuged at 12,000 g for 10 minutes at 4 ° C to precipitate RNA. The supernatant was carefully removed, the precipitate was gently washed 2-3 times with 1 ml of fresh 75% ethanol, dried at room temperature for 2 to 3 minutes and dissolved in 50 μl of RNase-free water. Stranded PolyA+ RNA libraries were prepared at Majorbio (Shanghai, China) with in-house kits. A total of 12 qualified cDNA libraries were constructed and were sequenced on the Illumina HiSeq XTen System (Illumina Inc., San Diego, CA).