GSM7770319: BMSC-shMETTL3-2-3; Homo sapiens; RNA-Seq
Total RNA was isolated from BMSC cells using an RNA Extraction Kit following the manufacturer's instructions (RX112, Vazyme, Nanjing, China). Experimental steps: 1. MRNA isolation and fragmentation -take 500ng totalrna, adjust the volume to 50ul with water, add 50ul mrnacapturebeads and mix well -place it on the PCR instrument at 65 ℃ for 5min, and then place it at room temperature for 5min -put it on the magnetic rack for 5min and remove the supernatant -remove it from the magnetic frame, add 200ul wash buffer and mix well -put it on the magnetic rack for 5min and remove the supernatant -remove it from the magnetic frame, add 50ul trisbuffer and mix well -place it on the PCR instrument at 80 degrees for 2min and 25 degrees hold -add 50ul beadbinding buffer, mix well, and place at room temperature for 5min -put it on the magnetic rack for 5min and remove the supernatant -remove it from the magnetic frame, add 200ul wash buffer and mix well -put it on the magnetic rack for 5min and remove the supernatant -add 19ul frame / prime buffer and mix well -put it on the PCR instrument at 94 degrees for 3min and 4 degrees for hold -put it on the magnetic rack for 5min, take out 16ul to a new EP tube, and prepare 0.12mg/ml actinomysin D solution 2.Double stranded cDNA --add 26ul of 1st strand buffer, 22ul of 1st strand enzyme mix and 1ul of actinomycin D (0.12 mg / ml) into 16ul supernatant and mix well -put it on the PCR instrument, 25 degrees for 10min, 42 degrees for 15min and 70 degrees for 15min -add 25ul of 2nd strand buffer 2 (with dUTP) and 15ul of 2nd strand enzyme super mix into the reaction product of the previous step, and mix well -put it on the PCR instrument, 16 degrees for 30min and 65 degrees for 15min 2. Add a connector -add 25ul rapid ligation buffer3, 5ul rapid DNA ligase2, 2 UL connector, 3ul water, mix well, place it on the PCR instrument for 20 degrees and 15min; -add 45ul DNA clean beans, mix well, and place at room temperature for 10min; -place the magnetic rack at room temperature for 5min, remove the supernatant and do not move the beads; -add 200 UL of 80% ethanol for 30 s at room temperature, remove the supernatant and repeat once; -dry at room temperature for 10min; -add 53ul water, remove it from the magnetic frame, fully mix it, and place it at room temperature for 5min; -place it on the magnetic frame and take the supernatant 50ul into a new pipe. -add 35ul DNA clean beans, mix well, and place at room temperature for 10min; -place the magnetic rack at room temperature for 5min, remove the supernatant and do not move the beads; -add 200 UL of 80% ethanol for 30 s at room temperature, remove the supernatant and repeat once; Dry at room temperature for 10min; -add 22ul water, remove it from the magnetic frame, fully mix it, and place it at room temperature for 5min; -place it on the magnetic frame and take the supernatant 19ul into a new pipe. 3. PCR enrichment -add 19uldna, 2.5ul i7 primer+2.5ul i5 primer, 25ulhifi amplification mix and 1ulheat labile UDG successively; -37 ° 10min, 98 ° 30s, 15 cycles (98 ° 10s, 60 ° 30s, 72 ° 30s), 72 ° 5min, 4 ° hold; -add 45ul amurebeads, mix well, and place at room temperature for 15min; -place the magnetic rack at room temperature for 10min, remove the supernatant and do not move the beads; -add 200 UL of 80% ethanol for 30 s at room temperature, remove the supernatant and repeat once, -dry at room temperature for 10min; -add 33ul water, remove it from the magnetic frame, fully mix it, and place it at room temperature for 5min; -place it on the magnetic frame and take the supernatant 30ul into a new pipe. 4. Quantification: qubit quantitative RNA library.