GSM4041593: K562_rep1_Red-C; Homo sapiens; OTHER
In Red-C experiments, cells were fixed with 1% formaldehyde, nuclei were isolated, DNA was fragmented with a restriction enzyme NlaIII, and the ends were blunted and A-tailed. RNA 3' ends were ligated with a bridge adapter containing a biotinylated nucleotide followed by ligation of the opposite ends of the bridge with DNA ends in a spatial proximity. RNA-DNA chimeras were purified and excessive DNA was cut off with MmeI restriction enzyme whose recognition site is in the bridge. After biotin-pull down, reverse transcription was initiated from the bridge with template switching at the 5' end of RNA (SMART technology) allowing for incorporation of a custom Illumina adapter. Finally, another Illumina adapter was ligated to DNA ends and the chimeras were amplified and paired-end sequenced on an Illumina platform. In the control experiment without DNA ligase, water was added instead of DNA ligase at the proximity ligation step. In the control experiment with RNase treatment, purified RNA-DNA chimeras were treated with RNase A prior to MmeI digestion. In RNA-seq experiments, we isolated total cellular RNA from K562 cells using RNeasy Mini Kit (Qiagen) followed by removal of ribosomal RNA using Ribo-Zero Gold rRNA Removal Kit (Human/Mouse/Rat) (Illumina). Red-C uses custom oligos and adapters resulting in libraries ready for sequencing on the Illumina platform. For RNA-seq we prepared strand-specific libraries using NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB).