GSM7235322: RDD 7SK Rep1; Drosophila melanogaster; OTHER
Dual-crosslinked cells were subjected for cell lysis and nuclei lysis. The lysed nuclei are fragmented into smaller complexes by sonication. Then the ncRNA of interest and its associated complexes are purified by hybridization using biotinylated antisense probes of a target ncRNA. And the purified complexes are immobilized on streptavidin C1 beads via high affinity with biotin, the excess unbound sites of beads are specifically blocked with denatured biotins to avoid the adsorption of biotinylated bridge-linker by streptavidin beads in later procedure of proximity ligation. Chromatin DNA contacts of complexes on beads are captured by incorporated biotinylated linkers between DNA sites during proximity ligation. Finally the ligated products are subjected for Tn5 tagmentation and enriched by streptavidin M280 beads for sequencing to obtain the specific RNA associated chromatin DNA–DNA interactions. The ligated DNA was fragmented by using Tn5 transposase. Then the fragmented DNA contained RDD-linker were captured with Streptavidin beads and used for PCR amplification. These PCR products were then performed double size-selection with AMPure beads to achieve the DNA fragments with size of 300-600 bp, then subjected for Illumina NovaSeq 6000 with paired-end sequencing at read lengths of 150 bp.