GSM5217189: HEK293T mouse IgG RIP ProtoScriptII small RNA rep1; Homo sapiens; ncRNA-Seq
To purify <200nt small RNAs, PureLink miR isolation kit (Invitrogen #K157001) was used with 1E7 starting cells or 50-100 μg total RNAs.Briefly, 1-5 μg purified small RNAs were immunoprecipitated with 5 μg anti-m1A antibody (MBL #D3453, or Abcam #ab208196) or corresponding IgG controls (mouse normal IgG – Millipore #12-371, or rabbit normal IgG – Millipore #12-370) conjugated on Protein G Plus Agarose (Pierce #22851) in the presence of SUPERaseIn RNase Inhibitor (Invitrogen #AM2694) at 4°C for 3 hours. Post binding, resin was washed with 1X IPP buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% NP-40) twice, low salt buffer (10 mM Tris-HCl, pH 7.4, 75 mM NaCl, 0.1% NP-40) once, high salt buffer (10 mM Tris-HCl, pH 7.4, 200 mM NaCl, 0.1% NP-40) once and TEN buffer (10 mM Tris-HCl, pH 7.4, 1 mM EDTA, 0.05% NP-40) twice. To elute bound RNAs, 3 mg/ml free 1-methyladenosine (Cayman Chemical #16937) in 1X IPP buffer was used to compete and displace m1A-containing RNAs at room temperature under rotation for 1 hour with a total number of two elutions. The eluted RNAs were further purified by acidic phenol-chloroform method and dissolved in water. Small RNA-seq library preparation was performed using NEBNext Small RNA Library Prep Set for Illumina (NEB #7330). The resulting amplified libraries were size selected on 8% TBE polyacrylamide gel to enrich for insert of 15-50 nucleotides long RNA.