GSM2732696: RBS2 CLIP-seq 2; Homo sapiens; OTHER
The RNA-protein complexes associated to RBS1, RBS2 or the TAP polypeptide were isolated as in the tandem affinity purification procedure  (IgG sepharose resin, calmodulin agarose resin) until the second purification step. After washing the calmodulin resin with the associated complexes, beads were incubated with 500 µl of Proteinase K Buffer (10 mM Tris-HCl pH 8, 100 mM NaCl, 0.5% SDS, 2 mM EGTA, 1 mM EDTA, proteinase K 0.2 mg/ml) at 65°C, during 2 h with gentle rocking. The elution products are recovered by gravity flow. RNAs were then extracted with phenol-chloroform, ethanol precipitated and resuspended in 20 µl of H20. Each RNA preparation was monitored using the Agilent 2100 Bioanalyzer (Agilent technologies). RNA (about 100 ng) obtained from two independent biological replicates of each sample (RBS1, RBS2 and TAP) were used to generate cDNA libraries using the TruSeq Stranded mRNA Sample Prep Kit v2 (Illumina) according to the manufacturer's protocol, but omitting the polyA+ selection step. All purifications steps were done using AMPure XP beads (Beckman Coulter). cDNA libraries were subjected to Illumina adapter ligation with TruSeq Universal Adapter 5’AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT3’ and TruSeq Adapter, Index 5’GATCGGAAGAGCACACGTCTGAACTCCAGTCACATCTCGTATGCCGTCTTCTGCTTG3’ and were sequenced in an Illumina HiSeq2000 sequencer. Reads length were 50 bp in a single-read run cycle. CLIP-Seq
GEO Accession
GSM2732696