GSM5940167: priB_TriC_Spt5_IgG1poolcapture_ctrl_clone1_rep1; Mus musculus; OTHER
TriC was performed as described (Downes et al 2022 with modifications desribed in Costea, Schoeberl et al 2022). In short, 10x10^6 or 15x10^6 (0h) cells were crosslinked with 2% HCHO for 8 min at room temperature. After quenching with 0,133M glycine, cells were lysed and nuclei isolated. Next, nuclei were lysed followed by NlaIII digestion (overnight), T4-DNA-ligation (overnight), Proteinase K digestion (overnight) and genomic DNA extraction. DNA was sonicated on Covaris S2 sonicator. TriC library was prepared of 3x2µg sonicated DNA using NEBUltraII and barcoding PCRs were performed using Nextflex unique dual index barcode sequences. For capture using reagents of the Roche HyperCap target enrichment kit and mouse Cot-1, 1-2µg of libraries were 2x (72h, 24h) hybridized to 2.9nM of individual biotinylated capture probes, followed by streptavidin pull down and PCR amplification using P5 and P7 (Illumina) primer sequences. Samples were multiplexed for sequencing on NextSeq or NovaSeq (Illumina) platforms