GSM5025898: WT2_iTreg_DFMO; Mus musculus; ATAC-seq
In vitro differentiated T-cells were sorted for live cells and stored in Bambanker freezing media (Thermo Fisher Scientific) at -80°C until further processing. Prior to library preparation, cells were thawed at 37  °C and washed with PBS. Cell pellets were lysed and tagmented in 1X TD Buffer, 0.2ul TDE1 (Illumina), 0.01% digitonin, and 0.3X PBS in 40ul reaction volume following the protocol described in (Corces et al., 2016). Transposition reactions were incubated at 37 °C for 30 min at 300 rpm. The DNA was purified from the reaction using a MinElute PCR purification kit (QIAGEN). The whole resulting product was then PCR-amplified using indexed primers with NEBNext High-Fidelity 2X PCR Master Mix (NEB). First, we performed 5 cycles of pre-amplification. We sampled 10% of the pre-amplification reaction for SYBR Green quantitative PCR to assess the number of additional cycles needed for final amplification. After purifying the final library with the MinElute PCR purification kit (QIAGEN), the library was quantified for sequencing using qPCR and a Qubit dsDNA HS Assay kit (Invitrogen). Libraries were sequenced on an Illumina NextSeq 550 system with paired-end reads of 37 base pairs in length.