GSM2564291: OverXTGIRTRep2 M1A IP; Homo sapiens; RIP-Seq
Briefly, 40 μl of protein-G magnetic beads were washed and resuspended in 200 μl of IPP buffer, and tumbled with 5 μl of affinity purified anti-m1A polyclonal antibody (Synaptic Systems) at room temperature for 30 minutes. RNA was added to the antibody-bead mixture, and incubated for 2 h at 4°C. The RNA was then washed twice in 200 μl of IPP buffer, twice in low-salt IPP buffer (50 mM NaCl, 0.1% NP-40, 10 mM Tris-HCl, pH 7.5), and twice in high-salt IPP buffer (500 mM NaCl, 0.1% NP-40, 10 mM Tris-HCl, pH 7.5), and eluted in 30 μl RLT (Qiagen). To purify the RNA, 20 μl MyOne Silane Dynabeads (Life Technologies) were washed in 100 μl RLT, resuspended in 30 μl RLT, and added to the eluted RNA. 60 μl 100% ethanol was added to the mixture, the mixture attached to the magnet and the supernatant discarded. Following two washes in 100 μl of 70% ethanol, the RNA was eluted from the beads in 10 μl H20. RNA was first subjected to FastAP Thermosensitive Alkaline Phosphatase (Thermo Scientific), followed by a 3’ ligation of an RNA adapter using T4 ligase (New England Biolabs). Ligated RNA was reverse transcribed either using SuperScript-III (Invitrogen) or using TGIRT-III (InGex), and the cDNA was subjected to a 3’ ligation with a second adapter using T4 ligase. The single-stranded cDNA product was then amplified for 9-12 cycles in a PCR reaction. Libraries were sequenced on Illumina Nextseq platforms generating short paired end reads, ranging from 25-55 bp from each end.
GEO Accession
GSM2564291