title: GSM9252552: CD4+T cells，WT-3; Mus musculus; ATAC-seq
library_construction_protocol: 1.Lysate the cells to extract the nuclei. 50,000 complete live cells were extracted. The number of live cells was detected using trypan blue, with 0.4% Trypan blue at 1 μ l and 9 μ l Cell suspension, stained for 3-5 minutes, microscopic examination technique.Add 50 μ l of pre-cooled RSB (add 0.1% NP40, 0.1% Tween-20, 0.01% digitalis soap) Invert three times and incubate on ice for 3 minutes.Add 1ml of pre-cooled RSB (add only 0.1% Tween-20), invert three times to mix well, and separate 500xg at 4°C. Wait for 5 minutes and discard the supernatant. 2.Transposase treatment Add 50 μ l of transposition mix, and incubate at 1000RPM at 37° C for 30 minutes using Qiagen MinElute PCR Purification Kit purifies DNA. DNA fragments were purified using the Zymo DNA Clean and Concentrator-5 Kit, with 10 μ l of 10 mm Tris Dissolve in buffer (PH8) and store at -20℃. 3.PCR amplification Search for the optimal number of amplification cycles. DNA fragments were purified using the Zymo DNA Clean and Concentrator-5 Kit, with 10 μ l of 10 mm Tris Dissolve in buffer (PH 8). Store at -20°C. 4. On-machine sequencing The library was sequenced through the illumina HiSeq/NextSeq platform after passing the detection. (1) clean data was obtained by removing the joint sequences and low-quality sequences using the Trimmomatic[2] program; (2) Use the Fastqc[3] program to count the data volume of clean data;(3) Use the bowtie2[4] program to align the clean data to the reference genome; (4) ATAC-SEQ-related quality control; (5) peak-calling on the genome using MACS2[4]; (6) Use the MEME[5] tool for motif detection of the binding peak; (7) Perform ChIPseeker[6] for peak annotation and screen the genes associated with the binding sites for functional enrichment; (8) Use DiffBind[7] for inter-sample difference analysis.