GSM9872196: Treg_Nat10kO_2; Mus musculus; RNA-Seq
For RNA-seq and Hi-C experiments, WT and Nat10-deficient CD4+ CD25+ Foxp3+ (GFP+) Treg cells from the lung tumors of the mice were purified by FACS. For CUT&Tag, cells were sorted enriched by ConA-magnetic beads, and then incubated overnight with antibody. The next day, beads were washed and incubated with anti-rabbit secondary antibody. Then the beads were washed and incubated with proteinA/G-Tnp transposome in Chitag. Next, cells were resuspended in 100 μL Tagmentation buffer (10 mM MgCl in Chitag Buffer) and incubated at 37 °C for 1 hr. The tagmentation was terminated by adding 1 μL of 10% SDS at 55 °C for 10 min. For HiC, the sorted cells were crosslinked with 1% formaldehyde and lysed in 1 ml lysis buffer. The resulting chromatin was then digested with NUB I (NEB) restriction enzymes at at 37 °C overnight. The DNA ends were blunted and labelled with biotin by Klenow DNA polymerase, followed by ligation using T4 DNA ligase (NEB). After reverse crosslinking, DNA was fragmented by sonication. For RNA-seq, RNA was extracted using the Direct-zol RNA MiniPrep kit (Zymo Research). A total amount of 1 μg RNA per sample was used as input material for the RNA sample preparations.For m3C-seq, RNA samples were subjected to alkaline hydrolysis for fragmentation. RNA fragments were dephosphorylated with antarctic phosphatase (NEB, USA) and then incubated in 1M aniline for cleavage. For RIP-seq, the RNA immunoprecipitation assay was carried out with GenSeq RIP kit (GenSeq Inc.) according to the manufacturer's instructions. rRNAs were removed by using GenSeq rRNA Removal Kit (GenSeq, Inc.). For RNA-seq, CD4+ CD25+ Foxp3+ (GFP+) Treg cells from the lung of mice bearing the intravenously inoculated B16F10 tumor cells were sorted by FACS (BD FACSAria III), each in three biological replicates. RNA was extracted using the Drect-zol RNA MiniPrep kit (Zymo Research). A total amount of 1 μg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext UltraTM RNA Library Prep Kit for Illumina (NEB) following manufacturer's recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (NEB). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3' ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 250-300 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter). Then 3 μL USER Enzyme (NEB) was used with size-selected, adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. For Hi-C, following the experimental condition and cell sorting strategy of Hi-C, 1 × 106 WT and Nat10-deficient CD4+ CD25+ Foxp3+ (GFP+) Treg were prepared per sample, each in two biological replicates. Hi-C was performed using the Hi-C 3.0 protocol as previously described(Lafontaine et al., 2021). In brief, the sorted cells were crosslinked with 1% formaldehyde for 10 min at 25 °C. The crosslinked cells were lysed in 1 ml lysis buffer (10 mM Tris-HCl, pH 8.0, 10 mM NaCl and 0.2% NP-40) supplemented with protease inhibitor cocktail (Millipore/Sigma) at 4 °C for 15 min. The nuclei were collected with 900 µl 1×CutSmart buffer (NEB) and then treated with 50 µl 0.5% SDS at 62 °C for 10 min, followed by adding 25 µl 10% Triton X-100 and 145 µl H2O to quench SDS. The resulting chromatin was then digested with NUB I (NEB) restriction enzymes at at 37 °C overnight. The reaction was stopped by standing on 65 °C for 20 min. The DNA ends were blunted and labelled with biotin by Klenow DNA polymerase in the presence of dCTP, dGTP, dTTP and biotin-14-dATP, followed by ligation using T4 DNA ligase (NEB). After reverse crosslinking, DNA was fragmented by sonication. The DNA fragments were then end-repaired and the biotinylated DNA fragments were captured using Dynabeads MyOne Streptavidin C1 beads (Invitrogen, Termo Fisher Scientifc). The DNA on beads was ligated to the Truseq adaptors and amplifed with PCR for library construction. DNA fragments of 200–500 bp were purifed from 1% E-gel and sequenced on Illumina HiSeq in paired-read mode with a read length of 150 nucleotides.