GSM6216030: TES6 input; Bos grunniens; RIP-Seq
The total RNA was isolated and purified using the TRIzol reagent . Bioanalyzer 2100 (Agilent, CA, USA) and NanoDrop 2000 (Thermo Scientific) with RIN number > 7.0 were used to evaluate the total RNA quality and quantity. poly (A) mRNA fractions are broken using divalent cations at high temperatures (RNA solution at 94°C for exactly 5 minutes using a thermocycler). Then, the fragmentation of broken RNA were incubated with m6A-Dynabeads (Anti-m6A, Synaptic Systems, Cat. No 202003) in an m6A-binding buffer (50 mM Tris-HCl pH7.4, 150 mM NaCl2, 1%NP-40, 2 mM EDTA), Allow the fragmented RNA to bind to the m6A-Dynabeads. Then washing of m6A-Dynabeads, Elution of m6A-positive RNA. Extraction and Cleanup of the RIP, Collecting m6A “enriched RNA”. We have generally utilized 100 ng of RNA (100 ng of input and 100 ng of post m6A-IP positive fraction) for library construction utilizing Illumina TrueSeq Stranded mRNA. Afterwards, we implemented the paired-end sequencing on an Illumina HiSeq X10 system, using a suggested protocol from the manufacturer.