GSM9422682: CD8+ T cell 9-pahsa treated rep3; Mus musculus; ATAC-seq
The assay for transposase-accessible chromatin (ATAC) was performed based on the Omni-ATAC protocol for optimal signal-to-noise ratio in immune cells. Briefly, fresh or cryopreserved CD8+ T cells were counted and washed in cold PBS. A total of 50,000 to 100,000 viable cells were lysed in cold ATAC Lysis Buffer to isolate nuclei. The nuclei were then immediately subjected to tagmentation reaction using the Illumina Nextera Tn5 Transposase, which simultaneously fragments accessible genomic DNA and inserts sequencing adapters. The reaction was performed at 37°C for 30 minutes in a buffer containing Mg2+. Tagmentation was stopped by adding SDS and EDTA. The tagmented DNA was then purified using a MinElute PCR Purification Kit , eluting in a small volume of Elution Buffer or nuclease-free water. The purified DNA constituted the pre-amplified library. The library construction was initiated directly from the purified tagmentation product, as the Tn5 transposase had already inserted the core adapter sequences. The DNA was first subjected to a limited-cycle, dual-indexing PCR amplification (typically 8-12 cycles) using a high-fidelity DNA polymerase and Nextera-compatible i5 and i7 indexing primers. The PCR program included an initial extension step (72°C for 5 minutes) for full adapter repair, followed by standard cycling conditions (98°C denaturation, 60-63°C annealing/extension). The amplified libraries were size-selected using a double-SPRI bead cleanup to remove excessive primer dimers and large fragments, enriching for nucleosome-free (< 200 bp) and mononucleosome (~200-600 bp) fragments. Final library quality and size distribution were assessed using an Agilent Bioanalyzer (High Sensitivity DNA chip) or TapeStation, and concentration was quantified via qPCR . Libraries were normalized and pooled for paired-end sequencing on an Illumina platform