GSM1370401: IP_EV; Homo sapiens; ncRNA-Seq
Affinity purification of 5’ capped RNAs was performed by incubating nuclear RNA with GST-eIF4E. Nuclear RNA pulled down with GST-eIF4E was then incubated with 125 pmol of (CCCTAA)3 oligonucleotide in 5 mM Tris-HCl pH7.5, 0.5 mM EDTA and subsequently heated to 75°C for 5 min to denature the RNA secondary structures. NaCl was added to a final concentration of 1M and the sample was cooled down to 50°C over a period of 30 min. Then, 25ml of Dynabeads (10mg/ml) (Invitrogen) were added and the mixture was incubated at 50°C for 3 hours. After washing the beads, the RNA was eluted by adding 100 ml of 10 mM Tris-HCl pH8 and heating to 80°C for 3 min. Libraries were prepared starting from 100 ng of RNA using the PE-102-1002-Paired-end kit from Illumina (Illumina, San Diego, CA, US) according to manufacturer recommendations. Ligation products of around 250 bp were gel purified before PCR amplification (15 cycles). 100 ng of libraries were then normalized according to the Illumina DSN protocol using the Duplex-specific nuclease from Evrogen (JSC, Moscow, Russia). 12 PCR cycles were performed using PCR PE 1.0 and 2.0 primers to enrich DNA fragments. DSN libraries were loaded on a HiSeq 2000 (Illumina) at 6 pM and paired-end sequenced using the 100 bp protocol.
GEO Accession
GSM1370401