GSM6617930: DP-Rag1ko-CBEwt-Trav17-Right-3C-HTGTS-rep3; Mus musculus; OTHER
For 3C-HTGTS, mouse thymus and liver were removed, cells were filtered through nylon mech.10 million cells were cross-linked with 1% formaldehyde. Cell were lysed and digest with 200U MboI overnight at 37°C. The sample was ligated with T4 DNA ligase overnight at 16°C. Crosslinks were reversed by Proteinase K and RNase A prior to DNA extraction with 1:1 phenol-chloroform and precipitation with ethanol. The 10ug 3C DNA was sonicated to 300-500bp and was linearly amplified with a biotinylated primer. The biotin-labeled single-stranded DNA products were enriched with streptavidin C1 beads, and followed by 3' ends ligation with the bridge adaptor. The adaptor-ligated products were amplified via nested PCR using a nested primer and an adaptor-complementary primer. And a final PCR for another 5-10 cycles with P5 and P7 was performed. After purification of the PCR products, the final libraries were sequenced on an Illumina NovaSeq 6000 platform to obtain 150 bp or 250 bp paired-end reads.