GSM8697643: P14 CD8+ T cells sgBcor/Zc3h12a, Day14, Replicate1; Mus musculus; ATAC-seq
Control, BCOR-deficient, ZC3H12A-deficient and BCOR/ZC3H12A-deficient P14 cells were transferred into Cas9+ B6 mice that were infected with LCMV Clone 13 one day before cell transfer. Fourteen days after transfer, Thy1.1+ or GFP+ P14 cells from spleen were sorted by a S3e cell sorter with purity > 98%. Cells were resuspended and washed with ice cold PBS for 2 times before permeabilization, and Tn5 tagmentation. To permeabilize cells, the cell pellets were resuspended in 50 μl of ATAC-seq RSB containing 0.1% IGEPAL-630, 0.1% Tween-20, and 0.01% digitonin and incubated on ice for 10 min. Afterwards, 1 ml of ATAC-seq RSB containing 0.1% Tween-20 (without NP40 or digitonin) was added, and the samples mixed by inverting the containing tubes for six times. Nuclei were then centrifuged for 10 min at 1000 r.c.f. in a pre-chilled (4 °C) fixed-angle centrifuge. Supernatant was removed, nuclei were incubated with Tn5 transposases and tagmentation buffer at 37°C for 30min (Novoprotein, N248). After tagmentation, stop buffer was added directly into the reaction. Samples were incubated at 55°C for 30min to terminate the tagmentation. Tagmentation products were purified by 2× VAHTS DNA Clean Beads. PCR was performed to amplify the library for 5 cycles using KAPA HiFi PCR Kit (Kapa Biosystems, kk2102) with the following PCR conditions: 72°C for 5min; 98°C for 3mins; and thermocycling at 98°C for 20s, 63°C for 30s. To determine the optimal amplification for each sample, 5ul PCR mix was diluted with 5ul H2O and combined with 2x SYBR qPCR supermix (Novoprotein, E096) to perform qPCR with following conditions: 98°C for 3mins; and thermocycling for 30 cycles at 98°C for 20s, 63°C for 30s and 72°C for 1mins. The additional number of PCR rounds (N) was determined as the qPCR cycle that reached the 1/3 maximum abundance on qPCR amplification curve. All samples were amplified for another N+3 cycles with the following PCR conditions: 98°C for 3mins; thermocycling at 98°C for 20s, 63°C for 30s and 72°C for 3min; and 72°C for 5mins for final extension. The library was sequenced on Illumina NovaSeq.