Chromatin isolation by RNA-purification sequencing assay (ChIRP-seq) for RAIN in papillary thyroid cancer cell line TPC1
Chromatin isolation by RNA-purification sequencing assay (ChIRP-seq) for RAIN in papillary thyroid cancer cell line TPC1
80*10^6 cells were collected for the ChIRP experiment. Cells were cultured in DMEM medium supplemented with 10% FBS and 1% P/S. Target RNA pull-down was carried out with two independent 3' BiotinTEG probe sets (EVEN and ODD) using Streptavidin Magnetic Beads. DNA was purified from 90% of ChIRP volume. Dry beads were resuspended in DNA-elution Buffer (500 mM NaHCO3, 1% SDS, 100 µg/ml RNaseA, and 0.1 U/ml RNaseH) and incubated at 37°C for 30 min while shacking. Beads were precipitated on a magnetic rack and a second round of elution was performed. Eluted DNA samples were incubated at 50°C for 45 min with Proteinase K. DNA was purified with PCR Purification Kit (Qiagen) following the manufacturer's instructions. Libraries were prepared with a starting DNA quantity of 150 pg with ThruPLEX DNA-Seq Kit (Takara Bio Ink)