title: GSM9374911: CD69-DP-Satb1KO-H3K4ME3-Rep3; Mus musculus; OTHER
library_construction_protocol: For ScRNA-seq, the  single-cell suspension was loaded onto a Chromium Single Cell Chip (Chromium Single Cell G Chip Kit, 10x Genomics, PN-1000120) according to the manufacturer's instructions by Guangzhou Yiyue Biotechnology Co., Ltd with the use of a Chromium Single Cell 3' GEM, Library & Gel Bead Kit v3.1 (10x Genomics, for co-encapsulation with barcoded Gel Beads at a target capture rate of about 10000 individual cells per sample. We lysed captured cells and released RNA was barcoded through reverse-transcription in individual single-cell gel beads in the emulsion (GEMS). In each droplet, complementary DNA (cDNA) was generated and amplified through reverse-transcription on a T100 PCR Thermal Cycler (Bio Rad) at 53 ℃ for 45 min, followed by 85 ℃ for 5 min and a hold at 4 ℃. Then, cDNA concentration and quality were assessed using Qubit Fluorometer (Thermo Scientific) and bioanalyzer2100 (Agilent), respectively. According to the manufacturer's introduction, scRNA-seq libraries were constructed and sequenced to a depth of 40000 reads per cell on illumina platform. For HiC-seq, one million cells were cross-linked at a final concentration of 1% formaldehyde and in room temperature for 10 min and quenched by 200 mM glycine for 5 min at room temperature. After washing with cold 1×PBS, cells were pelleted and kept at −80 ℃ until use. Subsequently, cross-linked cells were lysed to obtain nucleus, and chromatin was digested with DpnII and DdeI overnight at 37 ℃. DpnII and DdeI was inactivated at 65 ℃for 15 min, DNA fragment ends were biotinylated with biotin-dATP using Klenow large fragment (NEB, M0210V) for 90 min at 37 ℃, and the sample was diluted, and proximity ligated for 4 h at room temperature. DNA was treated with Lambda Exonuclease and Exonuclease I to remove unligated biotinylated ends. Cross-linked DNA was reversed by the addition of SDS, proteinase K, and allowed to incubate 4h at 55 ℃. DNA was purified by phenol-chloroform, followed by ethanol precipitation, and resuspended in 50 μl 1X TE. DNA was sheared to 300–500 bp by sonication subsequently, the ligated junctions were pulled down with Dynabeads MyOne Streptavidin C1 magnetic beads (Thermo Fisher Scientific, 65001). End repair, dA tailing, adapter ligation, and PCR amplification 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. The recommended sequencing volume gene is 100G every sample. For CUT&Tag, cells were ﬁxed by 0.5% v/v formalin in PBS for 10 min and quenched using 2.5 M glycine. The ﬁxed cells were permeabilized with TritonX-100 ; Then, cells were immobilized on a polylysine-coated coverslip instead of ConA beads and treated with different primary antibodies(anti-SATB1, Abcam 109122; anti-H3K4me3, Merck millipore 04-745; anti-H3K27ac, Abcam 4729; anti-CTCF, Merck millipore 07-729; anti-RAD21, Abcam 217678) and secondary antibodies (Goat anti-Rabbit IgG). Afterward, cells were incubated with pG-Tn5 transposase(Vazyme, S614-02) for 1 h at 37℃. Sample DNA was extracted using phenol-chloroform-isoamyl alcohol and ampliﬁed by PCR. All CUT&Tag libraries were sequenced on Illumina Nova Seq 6000 platform at PE150 mode. For ATAC-seq,  approximately 5 × 10⁴ cell pellets were washed once with cold phosphate-buffered saline (PBS). Cells were then lysed on ice for 3 minutes in 50 μL of ice-cold Lysis Buffer, which contained the following components: 10 mM Tris (pH 7.4), 10 mM NaCl, 3 mM MgCl₂, 0.1% NP-40, 0.1% TWEEN 20, and 0.01% Digitonin. Following lysis, the cell lysate was resuspended in 1 mL of ice-cold RBS-Wash Buffer (10 mM Tris [pH 7.4], 10 mM NaCl, 3 mM MgCl₂, and 0.1% TWEEN 20) and centrifuged at 4°C and 500 ×g for 5 minutes to pellet the cellular material. The tagmentation process was performed in 1× Tagmentation Buffer (comprising 10 mM Tris [pH 7.4], 5 mM MgCl₂, 10% dimethylformamide [DMF], 33% PBS, 0.1% TWEEN 20, and 0.01% Digitonin) with the addition of 100 nM Tn5 Transposase, and the reaction was incubated at 37°C for 30 minutes. Immediately after tagmentation, free DNA fragments were purified according to the standard protocol provided with the QIAquick PCR Purification Kit (QIAGEN, Cat. No. 28106, Germany). This purification step was followed by a final PCR amplification, which included 10–15 cycles using P5 and P7 primers. After post-amplification purification, the prepared libraries were subjected to sequencing on the Illumina NovaSeq 6000 platform to generate 150-bp paired-end reads (PE150). For ACC-seq,  thymocytes were dissociated into single cells. The cell suspension was then plated onto 0.3% gelatin-coated plates and incubated for 30 minutes at 37 ℃ in RPMI-1640 medium to remove adherent cells. A total of 3×10⁵ cells were divided into three aliquots and centrifuged at 500 rcf. For the native condition, cells were resuspended, washed with ice-cold PBS, permeabilized, and tagmented using Tn5 as in the standard ATAC-seq protocol. For the 1,6-Hex+Fix and Fix-only conditions, cells were centrifuged at 500 rcf for 5 minutes and washed once with PBS. In the 1,6-Hex+Fix group, cells were resuspended in PBS containing 10% (w/v) 1,6-hexanediol (Sigma-Aldrich, 240117) for 60 seconds at room temperature. Then, 16% formaldehyde (Invitrogen, 28906) was quickly added to the PBS to a final concentration of 1%, followed by fixation at room temperature for 10 minutes. The reaction was quenched with 0.6 M glycine (final concentration) for 5 minutes at room temperature. Cells were then washed once with ice-cold PBS and centrifuged at 500 rcf for 5 minutes. Subsequent steps were performed as in the ATAC-seq protocol. For SMART-seq2, 2000 cells sorted in 2x lysis buffer(0.4% triton x-100, 4U/ul RNase inhibitor) through BD FACS.The lysate samples were vortexed and centrifuged, and placed on a preheated PCR machine for heat-treatment. Then, Smart RT buffer and RT enzyme(SuperScript II reverse transcriptase) were added, and the reverse transcription reaction was carried out according to the program. Add 2x phanta enzyme and IS PCR primers to the cDNA product obtained in the previous step and carry out the amplification reaction according to the procedure. And take out a small amount of product and run electrophoresis gel for quality control. Quantitative purification of cDNA was performed using a DNA purification kit. The purified cDNA samples were added to Tn5 buffer and Tn5 enzyme, and the reaction was interrupted according to the program. After the reaction, mix Smart Amp buffer-2, Smart Amp enzyme-2, S5XX, N7XX for library construction, purify and send for testing. Illumina NovaSeq 6000 sequencing platform was usually used, and PE150 sequencing mode was adopted. Construction of libraries were described as the above extract protocol