GSM8193387: GDM-AGA-6; Homo sapiens; RNA-Seq
Extract total RNA from placental tissue using TRIzol reagent. Specifically: 1. Take the tissue from a -80 ℃ freezer, add a small amount of liquid nitrogen, grind it, take about 100 mg of the sample and place it in a 2 mL centrifuge tube. Add 1 mL of TRIzol reagent and lyse at room temperature for 10 minutes;  2. Add 0.2mL of chloroform to the lysis solution, cover the centrifuge tube tightly, vigorously shake the centrifuge tube for 15 seconds, let it stand at room temperature for 2-3 minutes, and centrifuge at 4 ℃ and 12000 rpm for 15 minutes. After centrifugation, the mixture can be divided into three layers, and RNA exists in the upper aqueous layer;  3. Carefully aspirate the upper aqueous phase into a new centrifuge tube, add 0.5mL of isopropanol, invert and mix well. Place on ice for 10 minutes, then centrifuge at 4 ℃ and 12000 rpm for 10 minutes. After centrifugation, RNA gel like precipitates can be seen attached to the side and bottom of the centrifuge tube. Discard the supernatant;  4. Wash the inner side of the tube wall thoroughly with 75% ethanol prepared by adding 1mL of DEPC water, and suspend and precipitate. Centrifuge at 4 ℃ and 12000 rpm for 3 minutes, discard the supernatant; Repeat twice;  5. Let the sample dry at room temperature for 2-3 minutes, add DEPC water to dissolve RNA, take a small amount of RNA solution for detection, and store the rest at -80 ℃. Using the NEBNext Ultra RNA Library Prep Kit for library construction, mRNA with polyA tails was first enriched using Oligo (dT) magnetic beads. The mRNA was randomly broken into small fragments using divalent cations in the fragmentation buffer. Short fragments of RNA were used as templates to synthesize the first and second strands of cDNA. Double stranded cDNA was purified using purified magnetic beads, followed by end repair, adding an A tail at the 3 'end, and connecting sequencing adapters. After connection, DNA magnetic bead purification and fragment selection were performed. Finally, PCR enrichment was performed to obtain the final cDNA library.