Illumina HiSeq 3000 sequencing; Cross-linking ligation and sequencing of hybrids (qCLASH) reveals an unpredicted miRNA targetome in melanoma cells
Cross-linking ligation and sequencing of hybrids (qCLASH) reveals an unpredicted miRNA targetome in melanoma cells
50 million cells in biological triplicates were UV cross-linked on ice at a wavelength of 250nm and 600J/cm2 in order to cross-link RNP complexes in living cells. The cell pellets were stored at -80Â°C until further use. The frozen cell pellets were thawn on ice and lysed using cell lysis buffer. Upon lysis, cells were treated with DNase and RNase T1, and the lysates were incubated overnight with mouse anti-Ago (2A8)-coated protein G Dynabeads. After immunoprecipitation, Ago-bound RNAs were phosphorylated. RNP complexes were incubated overnight with RNA ligase, followed by linker ligation. Upon complex elution from beads, the samples were treated with proteinase K. Nucleic acid was extracted using Phenol/Chloroform/Isoamyl Alcohol. After immunoprecipitation, Ago-bound RNAs were phosphorylated. RNP complexes were incubated overnight with RNA ligase, followed by dephosphorylation and 3' linker (miRCat-33) ligation. Upon complex elution from beads, the samples were treated with proteinase K. The RNA was phosphorylated and ligated to the Illumina TruSeq Small RNA Sample Prep Kits RA5 5' linker. RNA was reverse-transcribed using Illumina TruSeq Small RNA Sample Prep Kits RTP and amplified through 17 PCR cycles with Illumina TruSeq Small RNA Sample Prep Kits RP1 and the barcoded primer RPI1.