title: GSM8794308: ATAC.NC.rep1; Mus musculus; ATAC-seq
library_construction_protocol: Briefly, After washing the testicular tissue three times with PBS, the tissue digested with 1 mg/mL of type IV collagenase (ThermoFisher, 17104019) at 37°C for 15 min. After the tissues settle naturally, take the supernatant for culture in D-MEM/F-12 (Gibco, C11330500BT). After 48 hours, wash away the non-adherent cells with PBS, and then conduct immunofluorescence identification for the purity of LCs. ATAC-seq was performed using True Prep DNA Library Prep Kit V2 for Illumina (Vazyme, TD501). Cells were collected in PBS (2% BSA) and spun at 500 g at 4°C for 10 min. The pellet was resuspended in 50 μl lysis buffer and incubated at 4°C for 15 min and spun at 500 g at 4°C for 5 min. The supernatants were removed by carefully pipetting away from the pellets. For the transposition reaction, 10 μl 5×TTBL buffer, 5 μl TTE Mix V50 were combined and added to each pellet up to 50 μl. The samples were incubated at 37°C for 30 min followed by immediate purification using Beckman Beads. The PCR was set up in a 50 μl reaction volume using 24 μl of transposed DNA, 10 μl of 5×TAB, 5 μl PPM and 5 μl P5 and P7 primers in TruePrep Index Kit V2 for Illumina (Vazyme, TD202). PCR parameters were: 72°C for 5 min, 98°C for 30 s and 15 cycles of 98°C for 10 s, 60°C for 30 s and 72°C for 30 s. The libraries were purified using QIAGEN MinElute PCR purification kit (QIAGEN, Cat#28004) followed by Agencourt AMPure XP beads (Beckman Coulter, A63880). Library fragments ranging from 200 to 700 bp were enriched and the final elution volume was 30 μl. Libraries were subjected to paired-end 150 bp sequencing on NovaSeq platform at Cosmos Wisdom.