iPSC_RADICL-Seq_rep2.experiment
Nuclei were isolated, washed and genomic DNA was sheared by controlled DNase I digestion. After binding of nuclei to Ampure beads, genomic DNA was end-repaired and dA-tailed. During dA-tailing, RNA is treated with RNase H. After washes, nuclei were mixed with RADICL-seq adapter and T4 RNA Ligase 2-truncated KQ for ssRNA ligation. Subsequently, excess of adapter was washed away and nuclei were resuspended in T4 DNA ligase solution for ligation of RADICL-seq adapter to genomic DNA. RNA- and DNA-ligated RADICL-seq adapter molecules were first reverse-crosslinked by Proteinase K digestion at 65-degree, then precipitated and used for library preparation. The RNA ligated to the RADICL-seq adapter was first reverse transcribed with SuperScript IV and then converted into double stranded DNA by second strand synthesis. RADICL-seq adapters ligated only with RNA were neutralised by ligation with hairpin molecule with overhang A. Newly formed dsDNA molecules containing RADICL-seq adapter were first digested with EcoP15I fro generate paired-end tags, ligated with sequencing linkers and then affinity-purified by Streptavidin-conjugated beads. Recovered dsDNA was PCR amplified to generate libraries for Illumina sequencing and size-selected using a 6% native polyacrylamide gel.