GSM8850453: mT-2; Mus musculus; ATAC-seq
In brief, the sample was transferred from DMEM+10% FBS to a pre-chilled 2 ml Dounce (Sigma-Aldrich, USA) containing 1 ml of cold 1×homogenization buffer (HB) and thawed for 5 min. The sample was homogenized using an "A" loose pestle until resistance subsided (approximately 10 strokes), followed by further homogenization with a "B" tight pestle for an additional 20 strokes. The homogenate was filtered using a 70 µm Flowmi (Biosharp, China) strainer during transfer to a pre-chilled 2 ml LoBind tube (Eppendorf, USA). Nuclei were pelleted by centrifugation for 5 min at 4 °C and 350 RCF in a fixed-angle centrifuge (Eppendorf, USA). Aspirate the supernatant and discard until a volume of 50ul of liquid remains in the tube. The nuclei were gently resuspended in a total volume of 400 µl of 1×HB. An equal volume (400 µl) of 50% Iodixanol Solution (Sigma-Aldrich, USA) was added and thoroughly mixed by pipetting. A layer of 600 µl of 30% Iodixanol solution was carefully added underneath the 25% mixture, followed by a layer of 600 µl of 40% Iodixanol solution under the 30% mixture. The layered mixtures were then centrifuged for 20 min at 4 °C and 3,000 RCF in a pre-chilled swinging bucket centrifuge with the brake switched off. Care was taken to handle the tubes gently to avoid disturbing the gradient. The nuclei band was collected using a 200 µl volume and transferred to a fresh tube. The nuclei were then diluted by adding 500 µl of ATAC-RSB-Tween Buffer. The nuclei were enumerated using acridine orange/propidium iodide (AO/PI) (Sigma-Aldrich, USA) staining at a 1:1 ratio of AO/PI to the sample, and an automated cell counter hemocytometer (Countstar, China). Subsequently, 50,000 nuclei were transferred into a 1.5 ml LoBind tube containing 1000 μl of ATAC-RSB-Tween Buffer. The nuclei were centrifuged for 10 min at 500 RCF at 4 °C in a fixed-angle centrifuge. After discarding the supernatant, the nuclei were resuspended in 35 μl of ddH2O and transferred into an EP tube. ATAC-seq library preparation was conducted using the TruePrep DNA Library Prep Kit V2 for Illumina TD501(Vazyme, China). A volume of 15 μl of Tn5 reaction mix (10 μl of 5×TTBL and 5 μl of TTE Mix V50) was added following the kit instructions and thoroughly mixed. The mixture was incubated at 37 °C for 30 min. The resulting product was purified using a MinElute column (MinElute PCR Purification Kit, Qiagen, Germany). The PCR reaction setup included the purified fragmented DNA (24 μl), 5×TAB (10 μl), PPM (5 μl), N5** (5 μl), N7** (5 μl), and TAE (1 μl). PCR amplification was carried out according to the recommended protocol in the manual. The number of cycles was determined based on pre-experiment qPCR, set at 12 cycles, calculated as the cycle number at which the curve reaches the plateau phase in qPCR plus 5. Subsequently, the DNA product was purified again using a MinElute column (MinElute PCR Purification Kit, Qiagen, Germany). DNA sorting was performed using VAHTS DNA Clean Beads (N411 Vazyme) according to the 0.55×/1.0×scheme. Fragmentation analysis of the DNA was conducted using a Cartridge Kit (BiOptic, Taiwan). Finally, the libraries were sequenced using Illumina NovaSeq 6000 sequencing.