GSM8229349: KTC1 cells,Empty_vector-2,rep2; Homo sapiens; RNA-Seq
Add 200ul chloroform for every 1mlTRIzol, swirl and mix for 30 seconds, and let stand at room temperature for 3 minutes; Then centrifuge (4℃, 12000g×15 min) and slowly draw the upper water phase to the new 1.5ml EP tube. Add 500ul isopropyl alcohol for every 1ml of TRIzol, mix upside down, and let stand at room temperature for 10 minutes; After centrifugation, the supernatant was discarded (4℃, 12000g×10 min) to obtain RNA precipitation: the EP tube was inverted on the toilet paper and placed in a ventilated place to dry the RNA precipitation. Every 1ml of TRIzol was added with 1ml 75% ethanol, and then blown and mixed well. After centrifugation, discard the supernatant (4℃, 7500g×5 minutes), reverse the EP tube on the toilet paper, and place it in the fume hood to dry. Add nuclease-free water and mix well. Then heat at 55-60℃ for 10 minutes. The concentration and purity of RNA were determined by ultraviolet spectrophotometer to determine the light absorption of total RNA at 260nm and 280nm wavelengths. Total RNA was extracted from the samples by Trizol reagent (Invitrogen) separately. The RNA quality was checked by Agilent 2200 and kept at −80°C. The RNA with RIN (RNA integrity number) > 7.0 is acceptable for cDNA library construction. The cDNA libraries were constructed for each RNA sample using the TruSeq Stranded mRNA Library Prep Kit (Illumina, Inc.) according to the manufacturer's instructions. Generally, the protocol consists of the following steps: Poly-A containing mRNA was purified from 1ug total RNA using oligo(dT) magnetic beads and fragmented into 200-600 bp using divalent cations at 85℃ for 6 min. The cleaved RNA fragments were used for first- and second-strand complementary DNA (cDNA) synthesis . dUTP mix was used for second-strand cDNA synthesis, which allows for the removal of the second strand. The cDNA fragments were end repaired, A-tailed and ligated with indexed adapters. The ligated cDNA products were purified and treated with uracil DNA glycosylase to remove the second-strand cDNA. Purified first-strand cDNA was enriched by PCR to create the cDNA libraries. The libraries were quality controlled with Agilent 2200 and sequenced by NovaSeq 6000 on a 150 bp paired-end run.