GSM8117409
Around 20 million mouse cells were crosslinked with 1% formaldehyde. Nuclei were isolated, washed, and genomic DNA was sheared by controlled DNase I digestion. 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, nuclei were resuspended in T4 DNA ligase solution for ligation of RADICL-seq adapter to genomic DNA. The nuclear membrane was disrupted by sonication and immunoprecipitation was performed by using anti-H3K27me3 antibody with RNA-adaptor double-strand-DNA chimeric molecules. In parallel, we kept ten percentage of RNA-adaptor double-strand-DNA chimeric molecules and prepared libraries without antibody selection as the Input sample. RNA- and DNA-ligated adapter molecules were first reverse-crosslinked by Proteinase K digestion at 65°C, 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. Newly formed dsDNA molecules containing RADICL-seq adapter were first digested with EcoP15I for 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.