GSM7846914: ATAC-seq cortex Sham 2h rep3; Mus musculus; ATAC-seq
The mice were anesthetized and underwent transcardial perfusion with 10ml of isotonic saline. The ipsilateral cortex surrounding the injury site was swiftly dissected and preserved in pre-cooled DMEM supplemented with 10% FBS. The nuclei were counted using acridine orange/propidium iodide (AO/PI) staining (at a 1:1 ratio of AO/PI to sample) and an automated cell counter hemocytometer (Countstar Rigel S5). Then, 50,000 nuclei were transferred into a 1.5 ml LoBind tube containing 1000 ul of ATAC-RSB-Tween Buffer. The nuclei were centrifuged for 10 minutes at 500 RCF at 4°C in a fixed-angle centrifuge. The supernatant was discarded from the centrifuged product, and the nuclei were resuspended in 35 ul of ddH2O and transferred into an EP tube. The ATAC-seq library preparation was performed using the TruePrep DNA Library Prep Kit V2 for Illumina TD501. A volume of 15 ul Tn5 reaction mix (10 ul of 5×TTBL and 5 ul of TTE Mix V50) was added according to the kit instructions and mixed well. The mixture was then incubated at 37°C for 30 minutes. The resulting product was purified using a MinElute column (MinElute PCR Purification Kit, Qiagen). The PCR reaction system was set up with the purified fragmented DNA (24 ul), 5×TAB (10 ul), PPM (5 ul), N5** (5 ul), N7** (5 ul), and TAE (1 ul). PCR amplification was performed following the recommended protocol in the manual. The number of cycles was determined based on the pre-experiment qPCR to be 12 cycles, calculated as the cycle number at which the curve reaches the plateau phase in qPCR plus 5. The DNA product was then purified again using a MinElute column (MinElute PCR Purification Kit, Qiagen). The DNA was sorted using VAHTS DNA Clean Beads (N411 Vazyme) according to the 0.55×/1.0× scheme. Fragmentation analysis of the DNA was performed using a Cartridge Kit (Bioptic). Finally, the libraries were sequenced using Illumina NovaSeq 6000 sequencing.