title: GSM9021724: Treg_nontargeting_control_2; Mus musculus; ATAC-seq
library_name: GSM9021724
library_construction_protocol: We adapted a previous protocol for gene editing using CRISPR/Cas9 ribonucleoprotein (RNP) complex nucleofection(Seki & Rutx, 2018) for application to primary mouse Tregs for editing at the Smarcc1 locus. We isolated CD4 T cells from pooled spleen and lymph nodes of 6-8-week-old Foxp3IRES-GFP mice and using magnetic negative selection using the Dynabeads Untouched Mouse CD4 Cells Kit (ThermoFisher Scientific, cat#11415D), resting cells for at least 30 min at 4°C prior to electroporation. We freshly prepared gRNA duplexes by combining crRNA (pre-designed validated crRNA, IDT) and tracrRNA oligos (IDT) at equimolar concentrations to a final duplex18 concentration of 40 μM, heating the duplex at 95°C for 5 min in a PCR thermocycler and cooling down at RT for 15 minutes before keeping on ice. For every reaction, we used 12x106 CD4 T cells, washed with Ca/Mg free PBS and spun down at 200G for 10 min at RT to remove FBS. During this spin, immediately prior to nucleofection, we assembled RNP complexes by thoroughly mixing 2.25 μl of 5 μg/μl TrueCut Cas9 Protein V2 (Thermo Fisher) with 5ul 40μM gRNA duplex and incubating at RT for 15 minutes. For each target, we used a pool of two RNPs carrying different gRNAs duplexes (assembled separately). Cells were resuspended in 100 ul of Electroporation Solution P4 (Lonza) with the pool of both targeting RNPs or with RNPs carrying control non-targeting gRNAs for 2 min at RT and transferred into Large Lonza cuvettes for nucleofection with a Lonza Amaxa 4D Nucleofector (Program DS137). 150 μl of pre-warmed complete T cell medium (RPMI-1640, 10% FBS, supplemented with L-Glutamine, NEAA, beta- mercaptoethanol, and 10mM HEPES) with pre-equilibrated cytokine (10ng/mL recombinant mouse IL7 and 2000 IU/mL recombinant human IL2) was added immediately afterwards and cells were rested for 1h at 37°C. Cells from each cuvette reaction were subsequently transferred to separate wells of a 12 well plate containing 1mL of pre-warmed complete medium, adding another 750 μl of media used to wash residual cells from cuvettes. After 2 additional hours of rest, cells were expanded with anti-CD3/anti-CD28 beads (Thermo Fisher) at a 1:1 ratio for 3 days. All cells in culture were transferred into separate Treg-depleted Foxp3DTR hosts, injected with DT (Sigma) at 20 ng/g mouse body weight for two consecutive days prior to transfer and again on the day following transfer. Cells were parked in vivo for 1 week prior to sorting 10,000 GFP+ Tregs from pooled spleen and lymph nodes for bulk ATAC-seq. 3 biological replicates passing quality-control were used for targeting conditions and 2 biological replicates passing quality-control for non- targeting condition Bulk ATAC-seq libraries were prepared using the ImmGen ATAC-seq protocol as described in Yoshida et al, Cell, 2019