title: GSM8397353: Cd24a+Pax9- anti-IgG Cut&Tag_rep2; Mus musculus; OTHER
library_construction_protocol: Sample1-2 Following euthanasia of mice and tooth extraction, dental pulp was subsequently obtained using a syringe needle to remove epithelial tissue while preserve the mesenchymal part. The collected specimens were then placed into 1.5ml centrifuge tubes, finely minced for 3min, and subjected to a 5min enzymatic dissociation in 0.5ml of 0.25% trypsin-EDTA (ThermoFisher, 25200072). Neutralization was achieved with 1ml of culture medium (DMEM basal medium (Gibco, C11995500BT) supplemented with 10% fetal bovine serum (Lonsera, S711-001)). The medium was further supplemented with 20μl of 5mg/ml DNase I (Roche, 10104159001) and thoroughly mixed. After straining through a 70µm strainer and resuspension in 1x PBS, flow cytometry was employed for the isolation of the targeted cell populations.  Sample3-6 Following euthanasia of mice and tooth extraction, dental pulp was subsequently obtained using a syringe needle to remove epithelial tissue while preserve the mesenchymal part. The collected specimens were then placed into 1.5ml centrifuge tubes, finely minced for 3min, and subjected to a 5min enzymatic dissociation in 0.5ml of 0.25% trypsin-EDTA (ThermoFisher, 25200072). Neutralization was achieved with 1ml of culture medium (DMEM basal medium (Gibco, C11995500BT) supplemented with 10% fetal bovine serum (Lonsera, S711-001)). The medium was further supplemented with 20μl of 5mg/ml DNase I (Roche, 10104159001) and thoroughly mixed. After straining through a 70µm strainer and resuspension in 1x PBS, flow cytometry was employed for the isolation of the targeted cell populations.  Sample7-11 Following euthanasia of mice and tooth extraction, PN3 molar tissue was embedded in OCT compound. Laser capture microdissection (LCM) was conducted following an established protocol using Cresyl violet staining (Merck, C5042) for enhanced visualization of cellular morphology on PEN Membrane slides (MMI, 50102). Five regions were dissected: "Center," "Direct Below," "Side," "Pax9 Positive," and "Pax9 Negative." Samples were collected on IsolationCaps (MMI, 50206). Sample12-27 Following euthanasia of mice and tooth extraction, dental pulp was subsequently obtained using a syringe needle to remove epithelial tissue while preserve the mesenchymal part. The collected specimens were then placed into 1.5ml centrifuge tubes, finely minced for 3min, and subjected to a 5min enzymatic dissociation in 0.5ml of 0.25% trypsin-EDTA (ThermoFisher, 25200072). Neutralization was achieved with 1ml of culture medium (DMEM basal medium (Gibco, C11995500BT) supplemented with 10% fetal bovine serum (Lonsera, S711-001)). The medium was further supplemented with 20μl of 5mg/ml DNase I (Roche, 10104159001) and thoroughly mixed. After straining through a 70µm strainer and resuspension in 1x PBS, flow cytometry was employed for the isolation of the targeted cell populations. Sample1-2 Cell counting and viability confirmation were performed using the Countess II Automated Cell Counter (Thermo Fisher, AMQAX1000). Samples were then utilized for single-cell RNA sequencing with the 10x Genomics system.By using laser capture microdissection and odified SMART-seq3 library preparation methods, the relationships between different tissue regions were compared. Sample 3-6 Smart-seq3 was conducted with slight modifications. Single cells were sorted into 96-well plates with lysis buffer, then incubated at 72°C. Reverse transcription was performed with a mix containing RNase inhibitor and Maxima H-minus RT enzyme at 42°C. PCR pre-amplification followed, using KAPA HiFi HotStart buffer and specific primers. Samples were purified using homemade PEG beads and quantified. Tagmentation was done with homemade Tn5 and the libraries were amplified using the TruePrep Index Kit V2. Finally, samples were pooled, purified, and sequenced with 150-bp paired end using NovaSeq6000 Sample 7-11 The LCM samples collected on the IsolationCaps (MMI, 50206) were checked before further lysis with 50µl of 4M Guanidine isothiocyanate (GuSCN) solution (Invitrogen, 15577-018). RNA precipitation was conducted by adding a 771µl buffer composed of 600µl anhydrous ethanol (Fisher Chemical, A995-4), 20µl of 1.5M sodium acetate (Invitrogen, AM9740), 1µl glycogen (Thermo Fisher, AM9516), and 150µl of DNase/RNase-Free Distilled Water (Thermo Fisher, 10977015). The mixture was then incubated at -80°C for 30min, followed by centrifugation at 4°C at 12,000g for 30min to recover RNAs. The obtained RNA was processed for library construction utilizing the smart-Seq3 protocol (2) with slight modifications: The lysis buffer comprised 0.5U/μl of recombinant RNase inhibitor (RRI) (Takara, 2313A), 0.5mM dNTP (Thermo Scientific, R0192), and 0.5μM Smart-seq3 oligo-dT primer (5′-biotin-ACGAGCATCAGCAGCATACGAT30VN-3′; HPLC) (Takara). Following lysis, 10μl of reverse transcription mix was added to each sample, comprising 25mM Tris-HCl, pH 8.0 (Thermo Fisher, AM9855G), 30mM NaCl (Thermo Fisher, AM9760G), 1mM GTP (Thermo Fisher, R1461), 2.5mM MgCl2 (Thermo Fisher, AM9530G), 8mM DTT (Thermo Fisher, 707265ML), 0.5U/μl RRI, 2μM of Smart-seq3 TSOs (5′-biotin-AGAGACAGATTGCGCAATGNNNNNNNNrGrGrG-3′; HPLC) (Takara), and 2U/μl of Maxima H-minus RT enzyme (Thermo Fisher, EP0751). This integrated approach facilitates the detailed transcriptomic profiling of discrete anatomical regions. Sample 12-27 After harvesting, cells were counted and centrifuged for 3 minutes at 600g at room temperature, followed by resuspension in 4ml PBS and another centrifugation. Cells were then resuspended in 2ml ice-cold NE1 buffer with Pierce Protease Inhibitor Tablets (Thermo Fisher, A32965) and left on ice for 10 minutes. After centrifugation at 1,300g for 4 minutes at 4°C, cells were resuspended in 2ml PBS and centrifuged again. Nuclei were resuspended in wash buffer to approximately 1 million cells/ml, frozen in cryogenic vials with 10% DMSO, and stored at -80°C. Upon thawing, nuclei were incubated with Concanavalin A-coated magnetic beads (Bangs Laboratories, BP531), and then with primary antibody (α-H3K4me3 rabbit polyclonal antibody (Diagenode, C15410003)) and negative control (guinea pig α-rabbit IgG antibody (Antibodies online, ABIN101961)) overnight at 4°C. Secondary antibody (guinea pig α-rabbit IgG antibody (Antibodies online, ABIN101961)) was added and incubated for 1 hour. pA-Tn5 transposon was then added, and tagmentation was performed for 1 hour at 37°C. DNA was extracted, amplified by PCR for 17 cycles, purified using VAHTS DNA Clean Beads (Vazyme, N411), and sequenced at 150-bp paired end by Novaseq6000