title: GSM8651716: Hippocampus, Light phase, Syt7 KO, vehicle, 30min, rep1; Mus musculus; RNA-Seq
library_construction_protocol: 1. Add 1 mL of TRIzol™ Reagent per 50 mg of hippocampual tissue to the sample. 2. Pipet the lysate up and down several times to homogenize. 3. Incubate for 5 minutes to permit complete dissociation of the nucleoproteins complex. 4. Add 0.2 mL of chloroform per 1 mL of TRIzol™ Reagent used for lysis, then securely cap the tube. 5. Incubate on ice for 2–3 minutes. 6. Centrifuge the sample for 30 minutes at 12,000 × g at 4°C. The mixture separates into a lower red phenol-chloroform, and interphase, and a colorless upper aqueous phase. 7. Transfer the aqueous phase containing the RNA to a new tube. 8. Add 0.5 mL of isopropanol to the aqueous phase, per 1 mL of TRIzol™ Reagent used for lysis. 9. Incubate at -80 ℃ for 30 mins. 10. Centrifuge for 30 minutes at 12,000 × g at 4°C. Total RNA precipitate forms a white gel-like pellet at the bottom of the tube. 10. Discard the supernatant with a micropipettor. 11. Resuspend the pellet in 1 mL of 75% ethanol per 1 mL of TRIzol™ Reagent used for lysis. 12. Vortex the sample briefly, then centrifuge for 5 minutes at 7500 × g at 4°C. 13. Discard the supernatant with a micropipettor. 14. Air dry the RNA pellet for 5–10 minutes. 15. Resuspend the pellet in 20–50 µL of RNase-free water.16. Deliver the samples to the company using dry ice. 1)There are two methods to treat total RNA. Oligo(dT)magnetic beads are used to select mRNA with poly A tail, or hybridize the rRNA with DNA probe and digest the DNA / RNA hybrid strand, followed by DNase I reaction to remove DNA probe. Then obtain the target RNA after purification. 2) Fragment the target RNA and reverse transcription to double-strand cDNA (dscDNA) by N6 random primer. 3) End repair the dscDNA with phosphate at5' end and stickiness 'A'at3'end, then ligate and adaptor with stickiness 'T'at3' end to the dscDNA. 4) Two specific primers are used to amplify the ligation product. 5) Denature the PCR product by heat and the single strand DNA is cyclized by splint oligo and DNA ligase. 6)Perform sequencing on prepared library.