GSM7910790: ctl, ip, 0h, 1; Mus musculus; RNA-Seq
After perfusion with ice-cold HBSS, cortical regions samples were dissected from mouse brain and stored at − 80°C until use. Tissues were homogenized in ice-cold lysis buffer (50 mM Tris, pH 7.4, 100 mM KCl, 12 mM MgCl2, 1% NP-40, 1 mM DTT, 1x protease inhibitor, 200 units/ml RNasin (Promega) and 0.1 mg/ml cycloheximide (Sigma-Aldrich) in RNase-free deionized H2O) 10% w/v with homogenizer (Precellys 24, PeQlab). Then homogenates were centrifuged at 10,000 g at 4°C for 10 min to remove cell debris. Supernatants were collected and removed: 50 μl were used as input analysis. Anti-HA Ab (1:100, Covance) was added to the supernatant with slow rotation at 4°C. Meanwhile, Dynabeads Protein G (Thermo Fisher Scientific) were equilibrated to the lysis buffer by washing three times. After 4 h of incubation with HA Ab, 100 μl pre-equilibrated beads were added to each sample and incubated overnight at 4°C. After 10-12 h, Dynabeads were washed with high-salt buffer (50 mM Tris, 300 mM KCl, 12 mM MgCl2, 1% NP-40, 1 mM DTT, 1x protease inhibitor, 100 units/ml RNasin and 0.1 mg/ml cycloheximide in RNase-free deionized H2O) three times for 5 min at 4°C. At the end of the washing, beads were magnetized and 350 µl RLT buffer from RNeasy Micro Kit (74004, Qiagen) was added to the beads. RNA was extracted followed with manufacturer's instructions. The library was prepared and sequenced by Novogene using the following methods. Preliminary quality control was performed on 1% agarose gel electrophoresis to test RNA degradation and potential contamination. Sample purity and preliminary quantitation were measured using Bioanalyser 2100 (Agilent Technologies, USA), which was also used to check the RNA integrity and final quantitation. For library preparation, the mRNA present in the total RNA sample was isolated with magnetic beads of oligos d(T)25. This method is known as polyA-tailed mRNA enrichment. Subsequently, mRNA was randomly fragmented and cDNA synthesis proceeds using random hexamers and the reverse transcriptase enzyme. Once the synthesis of the first chain is finished, the second chain is synthesized with the addition of an Illumina buffer. Together with the presence of dNTPs, RNase H and polymerase I from E.coli, the second chain will be obtained by nick translation. The resulting products go through purification, end-repair, A-tailing and adapter ligation. Fragments of the appropriate size are enriched by PCR, where indexed P5 and P7 primers are introduced, and final products are purified. The library was checked with Qubit 2.0 and real-time PCR for quantification and bioanalyzer Agilent 2100 for size distribution detection. Quantified libraries were pooled and sequenced on Illumina Novaseq 6000 platform, according to effective library concentration and data amount.