GSM7720015: RA5 shCONTROL; Homo sapiens; RNA-Seq
Total RNA was extracted using TRIzol™ Reagent (Invitrogen, 15596018). Then, RNA samples undergo the following process. (1) DNase I digestion: Add DNase I to the RNA sample, digest at 37 ℃ for 30 minutes to remove residual DNA from the sample, and purify and recover RNA using magnetic beads. (2) Remove ribosomal RNA and RNA fragmentation: Add 5XRT Buffer and ribosomal RNA probe to the sample RNA, perform temperature gradient reaction on RNA sample fragmentation and remove rRNA. (3) Synthesis of first strand cDNA: Add EpiScriptTM IV, RNase inhibitor, DTT, TSO, random primers to the RNA sample in step 2. Mix gently with the tube wall and centrifuge rapidly. React in a PCR machine according to the following procedure: 37 ° C, 90 minutes; 70 ° C for 15 minutes. (4) First round of PCR amplification: Add 2XpfuMax HiFi PCR ProMix and sequencing primers to the first strand cDNA sample, mix well, and perform amplification reaction in the PCR instrument. (5) Purify the PCR amplification product using EpiTM DNA Clean Beads at a ratio of 1X, and recover 300-400bp of DNA fragments using magnetic beads at a ratio of 0.65X/0.2X for the purified product. (6) Perform a second round of PCR amplification on the purified product, enriching 300-400bp of DNA fragments. (7) Quality inspection: Use Biotic Qsep100 Analyzer to inspect the library and check if the distribution of library size meets the theoretical size. EpiTM mini longRNA-seq kit (Epibiotek, E1802) was used for library preparation according to the instructions.