GSM6185841: Muscle cell, CD36 KD, rep1; Mus musculus; RNA-Seq
Total RNA was isolated and purified using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to manufacturer's instructions. The NanoDrop ND-1000 was used to determine the amount and purity of RNA in each sample (NanoDrop, Wilmington, DE, USA). The Agilent 2100 with RIN number > 7.0 was used to assess RNA integrity.Total RNA was isolated using TRIzol (Invitrogen, Carlsbad, CA, USA) reagent. The RNA was fully released after 5 min at room temperature and added with 100 μl chloroform. Centrifuge at 12000 r for 15 min, take about 160 μl of the upper aqueous phase, add an equal amount of isopropanol, and mix them thoroughly. The RNA precipitate was washed twice with 75% ethanol and dissolved in 20 μl RNase-free water, and the RNA concentration was measured by NanoDrop. The 260/280 ratio was used to determine the RNA quality. The cDNA was obtained by taking 1 μg total RNA and reverse transcription in accordance with the instructions of cDNA synthesis kit (#KR106-02, TIANGEN, Beijing, China). The reaction system of real-time fluorescence PCR was as follows: 1 μl cDNA template, 5 μl 2×FastStart Universal SYBR GreenMaster (#FP205-02, TIANGEN, Beijing, China), 0.5 μl each of forward and reverse primers, and 4 μl RNAse-free water. The PCR reaction procedure was as follows: predenaturation at 95 °C for 10 min; product amplification cycle reaction at 95 °C for 10 s; and heating at 60 °C for 10 s and 72 °C for 10 s for 40 cycles. The total RNA quantity of the samples was calibrated using GAPDH as an internal reference, and the RNA expression level of the target gene was calculated using the 2−ΔΔCt method. After the total RNA was qualified, eukaryotic mRNA was enriched by magnetic beads connected with Oligo(dT). The extracted mRNA was fragmented into short fragments by Fragmentation Buffer at Random. A cDNA strand was synthesized with Random hexamers using fragment mRNA as template. Then buffer solution, dNTPs, RNaseH and DNA Polymerase I were added to synthesize the two-stranded cDNA. AMPureXP Beads were purified double chain products. The STICKY end of DNA was repaired to flat end using T4 DNA polymerase and Klenow DNA polymerase activity. Base A was added to the 3'end and joints were added. Finally, PCR amplification was performed to obtain the final sequencing library.