GSM8230953: EACBEko-Rag1ko-DN-Trdv5-3C-HTGTS-rep1; Mus musculus; OTHER
For Hi-C, We performed with DN thymocytes from 3-4 Rag1-/- or EACBE-/- Rag1-/- mice per experiment or 10 million CD3-stimulated DP thymocytes from Rag1-/- mice per experiment. In brief, cells were crosslinked with a final concentration of 1% formaldehyde at room temperature for 10 min and quenched with glycine (final concentration 0.125 M) on ice for 5 min. Crosslinked cells were lysed and followed by adding 150U DpnII(New England Biolabs,R0543L) to digest the chromatin overnight at 37℃ with gently shaking. DpnII was inactivated at 65℃ for 20 min, DNA fragment ends were biotinylated with biotin-dATP, and the sample was diluted, and proximity ligated for 4-6 hours at room temperature. Crosslinked DNA was reversed by addition of SDS, proteinase K, and NaCl, and allowed to incubate overnight at 68℃. DNA was purified by phenol/chloroform, followed by isopropanol precipitation, and resuspended in 100ul 10Mm Tris-HCl,pH:8.0. DNA was treated with T4 DNA polymerase to remove unligated biotinylated ends and sheared to 300-500bp by sonication subsequently, the ligated junctions were pulled down with Dynabeads MyOne Streptavidin C1 magnetic beads. End-repair, dA-tailing, adapter-ligation, and PCR amplification with P5 and P7 prime were performed on biotinylated DNA fragments bound to beads. After purification, libraries were sequenced on an Illumina NovaSeq 6000 platform to obtain 150 bp paired-end reads. 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.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 ATAC-Seq, a total of 5 × 104 cell pellets were washed once with cold PBS, cells lysed on ice for 3 min in 50 μL ice-cold Lysis Buffer (10 mM Tris pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% NP-40, 0.1% TWEEN 20, and 0.01% Digitonin in DEPC H2O), resuspended in 1 mL ice-cold RBS-Wash (10 mM Tris pH 7.4, 10 mM NaCl, 3 mM MgCl2, and 0.1% TWEEN 20) and pelleted at 4 °C at 500 × g for 5min. Tagmentation was performed in 1 × Tagmentation Buffer (10 mM Tris pH 7.4, 5 mM MgCl2, 10% DMF, 33% PBS, 0.1%TWEEN 20, and 0.01% Digitonin) using 100 nM Tn5 Transposase for 30 min at 37 °C. Immediately, Free DNA was purified using QIAquick PCR Purification Kit (QIAGEN, 28106, GERMANY) according to the manufacturer's instructions. And a final PCR for 10-15 cycles of amplification with P5 and P7 primers was performed. After purification, libraries were sequenced on an Illumina NovaSeq 6000 platform to obtain 150 bp paired-end reads. For HTGTS-VDJ, DNA was extracted by DNA Isolation Mini Kit (Vazyme, DC102) and sonicated to about 500bp on a Qsonica Bioruptor Sonicator. Sonicated DNA was linearly amplified with a biotinylated primer  that anneals to sites of interest. Biotin-labeled single stranded DNA products were enriched with streptavidin C1 beads (65001, Thermo Fisher Scientific), and followed by 3' end ligation with the bridge adapter. The adapter-ligated products were amplified through nested PCR using a nested primer and an adapter-complementary primer . The detailed primers used in this study are also listed in Supplementary information, Table S2. And a final PCR for another 10-12 cycles of amplification with P5 and P7 primers was performed. After purification, libraries were sequenced on an Illumina NovaSeq 6000 platform to obtain 150 bp paired-end reads. Construction of libraries were described as the above extract protocol