GSM5566214: MRC5-INPUT; Homo sapiens; OTHER
40 million MRC5 cells were collected and fixed with 1% glutaraldehyde in PBS for 10 min at room temperature, reflection terminated by 1.25 mM Glycine for 5 min. Cells were resuspended in lysis buffer and sonicate in a 4°C water bath. Supernatant was collected, and diluted with hybridization buffer and appropriate volume of probes (TERC odd probes or Laz-probe), incubate at 37°C for 4 h with shaking. Streptavidin-magnetic C1 beads were blocked with 500ng/ul yeast total RNA and 1mg/ml BSA for 1 hour at room temperature, and resuspended with original volume of lysis buffer and added to the hybridization system, 37°C for 30 min with shaking. Streptavidin-magnetic C1 were washed five times with wash buffer.  For DNA elution, beads were resuspended in 3×original volume DNA elution buffer with RNaseA and RNase H, followed by reverse cross-linking, proteinase K digestion and phenol-chloroform extraction. DNA fragments were precipitated by ethanol in the presence of NaAc and glycogen. Eluted genomic DNA was subject to high-throughput sequencing. Libraries were prepared according to Illumina's instructions. Briefly, genomic DNA was precisely quantified with Qubit™ dsDNA HS Assay Kit (ThermoFisher, Q32854), fragmented with Covaris and extracted with Hieff NGS™ DNA Selection Beads (Yeasen, 12601ES56). Then, DNA was end-repaired, adapter-ligated and amplified with NEB Next® Ultra™ DNA Library Prep Kit for Illumina® (NEB, E7370), and purified with Hieff NGS™ DNA Selection Beads (Yeasen, 12601ES56). Libraries were sequenced on the Illumina HiSeq following the manufacturer's protocols. ChIRP-seq