GSM4580099: RNA-DNA SPRITE 108; Mus musculus; OTHER
Cells were lifted using trypsinization and were crosslinked in suspension at room temperature with 2mM disuccinimidyl glutarate (DSG) for 45 minutes followed by 3% Formaldehyde for 10 minutes to preserve RNA and DNA interactions in situ. After lysis of cells, nuclei were lightly sonicated. DNA was DNAse digested to an average size of approximately 150-1000 bp in length. Crosslinked complexes of interacting DNA, RNA, and protein were isolated on NHS beads, RNA and DNA ends were repaired, DNA was dA-tailed to generate a protruding 3' A-base for ligation of barcodes, RNA was ligated with a partially ssRNA-dsDNA adaptor for ligation of barcodes, and were then split-pool tagged with a series of barcodes over multiple rounds of split-and-pool tagging. A distinct RNA adaptor was ligated to RNA to flag a molecule as RNA rather than DNA. This adaptor contains a shared overhang that allows for simultaneous tagging of RNA and DNA during the split-and-pool tagging process. Reverse transcription was performed to convert RNA into cDNA. After split-and-pool tagging, crosslinks were reversed, the DNA/RNA was isolated, was amplified 8-12 cycles using standard Illumina library prep (dual index), and purified using SPRI beads and gel cut. Libraries were sequenced on a NovaSeq S4 chip (150x150 read length).