title: GSM7882809: Re_24h input rep1; Mus musculus; OTHER
library_construction_protocol: Total RNA was isolated from cerebral cortical peri-infarct tissue of sham, MCAO/R-0h, MCAO/R-6h and MCAO/R-24h mice and extracted with TRIzol reagent (TaKaRa) according to the manufacturer's instructions. 10μg total RNA was incubated with 5μl DNase I at 37°C for 20 minutes to remove DNA contamination. 5 µg of total RNA was used for digestion and the total RNA was chemically digested into fragments of approximately 200nt length. Approximately 1/10 of the fragmented RNA was isolated as an input control for further RNA-Seq. The remaining fragmented RNA was mixed with 3.5 µg of m6A antibody (Millipore) magnetic beads pre-mixed in IP buffer [50mM NaCl, 10mM Tris-HCl, pH7.5, 0.1%IGEPAL CA-630] at 4°C overnight. The eluted RNA was precipitated with 75% ethanol. PCR primers HT (Indexing Primer Set HT for illumina® V2-96-Takara) were used to transform the eluted fragment containing m6A (IP) and the untreated input control fragment into the final cDNA library according to the stark-specific library preparation. Average insert size of paired-end libraries was ∼150 bp.