GSM7871872: SS2TcAct_2; Mus musculus; RNA-Seq
Live cells were sorted using PI staining, then 5 × 104 cells were transferred to a low-binding centrifuge tube and resuspended in 50 μL of pre-chilled Lysis Buffer. After 20 times pipetting, cells were incubated on ice for 10 minutes for lysis. RNA-seq The RNA-seq library preparation was performed by a modified SMART-seq2 protocol49,51. Briefly, RNA was added into 4 µl elution mix made of 1 µl RT primer (10 µM), 1 µl dNTP mix (10 mM each), 1 µl RNase inhibitor (4 U/µl), and 1 µl H2O. Eluted samples were incubated at 72 °C for 3 min and immediately placed on ice. Each sample was added with 7 µl reverse transcription (RT) mix made of 0.75 µl H2O, 0.1 µl Maxima RNase-minus RT (Thermo Fisher Scientific, #EP0741), 2 µl 5x Maxima RT buffer, 2 µl Betaine (5 M, Sigma-Aldrich, B0300), 0.9 µl MgCl2 (100 mM), 1 µl TSO primer (10 µM), 0.25 µl RNase inhibitor (40 U/µl).The RT reaction was incubated at 42 °C for 90 min and followed by 10 cycles of (50 °C for 2 min, 42 °C for 2 min), then heat inactivated at 70 °C for 15 min. Samples were then amplified with an addition of 14 µl PCR mix made of 1 µl H2O, 0.5 µl ISPCR primer (10 µM), 12.5 µl KAPA HiFi HotStart ReadyMix (KAPA Biosystems, KK2602). The PCR reaction was performed as follows: 98 °C for 3 min, 22 cycles of (98 °C for 15 sec, 67 °C for 20 sec, 72 °C for 6 min), and final extension at 72 °C for 5 min.  The amplified cDNA product was purified using VAHTS DNA Clean Beads. Sequencing libraries were prepared using TruePrep DNA Library Prep Kit V2 for Illumina (Vazyme, TD503-02).  Primer sequences: RT primer (Sangon), 5'Biotin-AAGCAGTGGTATCAACGCAGAGTACTTTTTT TTTTTTTTTTTTTTTTTTTTTTTTVN; TSO primer (Sangon), 5'Biotin-AAGCAGTGGTATCAAC CAGAGTACAT/rG//rG//iXNA_G/; ISPCR primer (Sangon), 5'Biotin-AAGCAGTGGTATCAACGC AGA*G*T. ATAC-sequencing ATAC-seq were performed using TruePrep DNA Library Prep Kit V2 for Illumina according to the manufacturer's recommendation. Briefly, live cells were sorted using PI staining, then 5 × 104 cells were transferred to a low-binding centrifuge tube and resuspended in 50 μL of pre-chilled Lysis Buffer. After 20 times pipetting, cells were incubated on ice for 10 minutes for lysis. Subsequently, cells were pelleted by centrifugation at 4°C 500 g for 5 minutes, and suspended in a mixture consisting of 10 μL of 5× TTBL, 5 μL of TTE Mix V50, and 35 μL of ddH2O. The mixture was gently mixed by pipetting 20 times, and incubated at 37°C for 30 minutes for fragmentation. The products were purified using 2× VAHTS DNA Clean Beads, eluted with 26 μL of water, and collected for the subsequent amplification. 24 μl of the eluted samples were amplified with different combination of i7 index and i5 index primers. QPCR were performed to determine the cycle number for PCR amplification.0.55×VAHTS DNA Clean Beads was employed for size selection of the PCR product. Finally, 30 ng of each sample were pooled for subsequent second-generation sequencing, on the Illumina NovaSeq 6000 with read length of 150 bp.  Cut&Tag assay Foxp3-RFP+ iTreg were induced, and infected with NFAT-Flag-GFP lentiviruses on day 1.5 and day 2.5 of induction. On day 5, Foxp3-RFP+ MSCV-NFAT-Flag-GFP+ iTregs were sorted, then rested and restimulated for 2 day. Cut&Tag assay were performed using Hyperactive Universal CUT&Tag Assay Kit for Illumina (Vazyme Biotech Co.,Ltd) according to the manufacturer's recommendation. Briefly, 1 × 105 iTregs were collected to low-binding centrifuge tubes and washed. Nucleus isolation were performed by NE buffer for 10 min on ice, and cross-linked by 0.1% formaldehyde for 2 min, then stopped by glycine. Con A Beads were pre-treated with binding buffer, and then incubated with nuclei of iTreg for 10 min. Further, samples were incubated with anti-Flag M2 antibody (1:50) or without primary antibody (Mock) overnight at 4°C. Goat Anti-Mouse IgG secondary antibody (1:50) was added and incubated for 1 h. Cells were washed three times, and then incubated with pA/G-Tnp enzyme for 1 h. After incubation, samples were mixed with 5×TTBL and Dig-300 Buffer and incubated at 37°C for 60 min for fragmentation. DNA was extracted and purified, then amplified with different combination of i7 index and i5 index primers. QPCR was performed to determine the cycle number for PCR amplification.VAHTS DNA Clean Beads were employed for size selection of the PCR product. Finally, 30 ng of each sample were pooled for subsequent second-generation sequencing, on the Illumina NovaSeq 6000 with read length of 150 bp.