title: GSM5548384: DP-Satb1WT-ETS2p-3C-HTGTS-rep2; Mus musculus; OTHER
library_construction_protocol: For ChIP-Seq, DP thymocytes were perforated and dealed with MNase.Histone-DNA particles were precipitated with antibody. DNA were purified by PCR Purification Kit.For library preparation, DNAseq libraries were made using Tn5 DNA Library preparation kit. For ATAC-Seq, a total of 50,000 cell pellets were washed with PBS, cells lysed on ice for 3 minutes in 50 μL ice-cold Lysis Buffer, and resuspended in 1mL ice-cold RBS-Wash and pelleted at 4°C at 500 × g for 10 minutes. Tagmentation was performed in 1 × Tagmentation Buffer (10mM Tris pH 7.4, 5mM MgCl2, 10% DMF, 33% PBS, 0.1% TWEEN 20, 0.01% Digitonin) using 100nM Tn5 Transposase for 30 minutes at 37°C. DNA was purified using PCR Purification Kit according to the manufacturer's instructions. DNA was eluted into a volume of 20 μl and stored at -20°C prior to library preparation.For library preparation, PCR were performed using Kappa High-Fidelity DNA Polymerase with 1 cycle of 5 minutes at 72°C for gap filling. And a PCR for another 10-15 cycles with P5 and P7 was performed. After purification of the PCR products, the final libraries were sequenced on an Illumina HiSeq 2000 platform to obtain 150 bp paired-end reads. For 3C-HTGTS, in brief, 10 million cells were cross-linked with 1% formaldehyde. Cell were lysed and digest with 200U MboI overnight at 37°C. MboI was inactivated by adding 10%SDS at 37°C for 30 min . To reduce the SDS concentration, the solution was diluted with T4 ligase buffer containing 1% Triton X-100, followed by incubation at 37°C for 1h. T4 ligase was added to each sample and incubated for overnight at 16°C.Crosslinks were reversed and samples were treated with Proteinase K and RNase A prior to DNA extraction with 1:1 phenol-chloroform and precipitation with ethanol.The 3C libraries were sonicated to 300-500bp Sonicated DNA was linearly amplified with a biotinylated primer that anneals baits . 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 10-15 cycles with P5 and P7 was performed. After purification of the PCR products, the final libraries were sequenced on an Illumina HiSeq 2000 platform to obtain 150 bp paired-end reads. For 4C-Seq, chromatin was crosslinked for 10 minutes at room temperature in 1X PBS/10% FBS containing 4% formaldehyde. Crosslinking was blocked by glycine addition. Following cell lysis, nuclei pellets were resuspended in 1.2X Buffer2 (NEB), followed by SDS addition. The samples were incubated while shaking for 60 minutes at 37°C. TritonX-100 was added in order to quench SDS. Crosslinked chromatin was primarily digested using MboI . Ligation was performed in the presence of T4 DNA Ligase in diluted conditions. Chromatin reverse crosslinking was carried out using proteinase K ,phenol-chloroform and DNA were purified by ethanol precipitation. Secondary restriction was performed using NlaIII. Secondary ligation was carried out with T4 DNA Ligase in diluted conditions. Ligated DNA products were then extracted using phenol-chloroform with ethanol precipitation and purified.4C-seq library preparation was achieved by inverse PCR using 100 ng template DNA (for 10 reactions in total),. The viewpoint-directed inverse PCR primers carrying Illumina P5 or P7 sequencing adapters as below: Ets2 forward primer:5'- GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTgcccgggagcagcgtggatc-3';Ets2 veserce primer: 5'-TCTTTCCCTACACGACGCTCTTCCGATCTcgagcacttaggcgccatct-3'. For Hi-C, 5-10 million  DP thymocytes were crosslinked at 1% formaldehydeand and quenched by 200mM glycine for 5min at room terperature. After two washes with cold PBS,cells were pelleted and kept at -80°C until use. Subsequently, crosslinked cell were lysed and chromatins were digested with 150 U MboI overnight at 37 °C. Next, MboI was inactivated at 62 °C for 20min, fragmented ends were  biotinylated with biotin-dCTP using the Klenow large fragment for 60-90 min at 37 °C, and the sample was diluted and proximity ligated for 4 h at room temperature. Crosslinks DNA were reversed by the addition of SDS, ProteinaseK, and NaCl, and allowed to incubate overnight at 68 °C. DNA was purified by phenol/chloroform, followed by ethanol precipitation and resuspended in 100ul nuclease-free dH2O. DNA was treated with T4 DNA polymerase to remove unligated biotinylated ends and shared to 300-500bp by sonication Subsequently,The ligated junctions were pulled down with Dynabeads MyOne Streptavidin C1 magnetic beads.End-repair, A-tailing, adapter-ligation and PCR amplification reactions were performed on the biotinylated fragments bound to the beads. After purification of the PCR products, the final libraries were sequenced on an Illumina HiSeq 2000 platform to obtain 150 bp paired-end reads. For 5' RACE, the 1ug RNA was used for the reverse transcription (RT). The cDNA libraries were performed with oligo(dT)  primer by 5′Rapid amplification of complementary DNA ends(5'RACE) . During the reverse transcription reaction , A SMART oligonucleotide was ligated to  the 5'end of each cDNA . The TCR library is then prepared by touchdown PCR amplification using a set of primers: a forward primer to the adaptor and a reverse primer to the constant region of the TCR  . Touchdown PCR was performed under the conditions of 95°C for 3 min, followed by 15 cycles of 98 °C forc15 s, 65 °C(decrease 0.7°C/cycles) for 15 s and 72 °C for 1 min,  20 cycles of 98 °C for 15 s, 60 °C for 15 s and 72 °C for 1 min and one cycle 10 min at 72°C. PCR products, ranged from 300-700bp were gel purified by DNA Gel Purification Kit. Illumina paired end adapters were ligated to TCR libraries, followed by 2rd round PCR with Illumina P5/P7 adaptor primers under 30s at 95°C , 15s at 65 °C, 60s  at 72°C for 10-15 cycles , Plus a final extenaion for 10 min at 72°C. Ampure bead purified PCR product. Finally, the TCR libraries were sequenced on an Illumina HiseqXtenPE150 (2 × 150 nt). For Bulk RNA-Seq, the sorted DP cells were isolated using a TRIzol. The total RNA was quantified and qualified by Agilent 2100 Bioanalyzer and NanoDrop2000 .Subsequently, 1 μg total RNA was used for following library preparation. The poly(A) mRNA isolation was performed using Poly(A) mRNA Magnetic Isolation Module. First strand cDNA was synthesized using ProtoScript II Reverse Transcriptase(NEB) and the second-strand cDNA was synthesized using Second Strand Synthesis Enzyme Mix(NEB). The purified double-stranded cDNA were subjected to end repair, 3'-dA tailing and adapter ligation.Size selection of adapter ligated DNA were performed prior to PCR amplification. The ligated DNA was then amplified by 10-15 PCR amplification cycles with Illumina P5/P7 primers. The PCR products were cleaned up using beads, validated using an Qsep100 and quantified by Qubit3.0 Fluorometer. Libraries were a 2x150bp paired-end (PE) sequenced on a NextSeq 550 on an Illumina HiSeq instrumen For scRNA-Seq, thymocytes were isolated from thymus. Cell viability was assessed and the samples (cell viability >80%) were prepared for single-cell library construction. The single-cell library was constructed using the ChromiumTM Controller and ChromiumTM Single Cell 3' Reagent Version 2 Kit (10x Genomics, Pleasanton, CA) according to the manufacturer's instructions. The PCR products were then quality and quantitated by Agilent 2100 bioanalyzer and  QPCR. The final libraries were sequenced using the Illumina Hiseq 4000 (BGI-Shenzhen, China).