title: GSM8017912: 2D ES_1; Mus musculus; RNA-Seq
library_construction_protocol: 7. Place the column on 2 ml collection tube, add 400 µ l buffer Rb, centrifuge as described above, discard the filtrate and reuse the collection tube. 8. Place the column on the same 2 ml collection tube, add 600 µ l RNA wash buffer, centrifuge as described above, discard the filtrate and reuse the collection tube. 9. Maximum speed (13000) × g) Centrifuge for 2 min and completely dry the film 10. Transfer the column to a clean 1.5 ml RNase free microfuge tube. Add 50 µ L DEPC treated ddH2O, dissolve RNA, stand at room temperature for 2 min, and centrifuge at the maximum speed for 1 min. 11. Determine the concentration and electrophoretic quality inspection RNA. 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.(VAHTS® Universal V8 RNA-seq Library Prep Kit for Illumina(Cat.NR605),Vazyme)