title: GSM8862703: Knock-out 1; Mus musculus; ATAC-seq
library_construction_protocol: Stimulated CD4+T-cells from WT and Keap1-KO mice were harvested after 36 h. Cells were transferred to a 15mL or 50mL conical tube and centrifuged at 500 x g for 5 minutes. The media was aspirated, and cells were resuspended in 10mL of pre-warmed growth media, followed by the addition of 1:100 volume of 100x DNase buffer and DNase solution (250mM MgCl2 and 50mM CaCl2 in dH2O). Incubated the cells at 37°C for 30 minutes and centrifuged again at 500 x g for 5 minutes at 4°C, resuspended in 5mL ice-cold PBS (or 500µL for Eppendorf tubes), and 300,000 cells were quantified using a hemocytometer for cryopreservation. Cryopreserved cells were sent to Active Motif to perform the ATAC-seq assay. The cells were then thawed in a 37oC water bath, pelleted, washed with cold PBS, and tagmented as previously described (Buenrostro et al. 2013), with some modifications based on (Corces et al. 2017). Briefly, cell pellets were resuspended in lysis buffer, pelleted, and tagmented using the enzyme and buffer provided in the ATAC-Seq Kit (Active Motif cat# 53150). Tagmented DNA was then purified using the MinElute PCR purification kit (Qiagen), amplified with 10 cycles of PCR, and purified using Agencourt AMPure SPRI beads (Beckman Coulter). Resulting material was quantified using the iSeq (Illumina) and sequenced with PE42 sequencing on the NovaSeq 6000 sequencer (Illumina).