GSM8728185: EACBEwt-Rag1ko-DP-Tcrb-CBE3-3C-HTGTS-rep1; Mus musculus; OTHER
For 3C-HTGTS, mouse thymus 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.The detailed primers used in this study are also listed in Supplementary information, Table S1.  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 paired-end reads. For ChIP-Seq, Chromatin immunoprecipitation (ChIP) was performed on DN thymocytes from 3-4 Rag1-/- mice and 10 million anti-CD3 antibody-stimulated DP thymocytes from Rag1-/- mice per experiment. Cells were crosslinked with a final concentration of 1% formaldehyde on ice for 10 minutes and quenched with glycine (final concentration 0.125 M) on ice for 5 minutes. Cells were washed in 1x PBS once. The subsequent steps were carried out as ABclonal Sonication ChIP Kit (RK20258) with some modifications. After lysed and sonicated, chromosomal DNA was incubated with 5μg of specific antibody (anti-CTCF 07-729 from Millipore, anti-Rad21 Ab992 from Abcam) or isotype-matched IgG for 5 hours at 4°C, followed by addition of 15μl protein A/G magnetic beads for an additional 2 hours at 4°C. After washing and reversal of cross-linking, Immunoprecipitates place tube on magnet and collect supernatant to a new tube.The product was purified by PCR purification kit. Immunoprecipitated and input DNAs were quantified by real time PCR or sequenced on an Illumina NovaSeq 6000 platform to obtain 150 bp pair-end reads. Construction of libraries were described as the above extract protocol