Essential role of ThPOK autoregulatory loop in maintenance of mature CD4 T cell identity and function [ATAC-seq]

The transcription factor ThPOK is critical for homeostasis and differentiation of mature T helper cells. Overall goal of this experiment is to show divergent Chromatin pattern of ThPOK-dependent autoregulatory loop disrupted in CD4 T cells compared to WT. 5 × 10^4 flow sorted naïve CD4 cells from WT or CD4lo cells from OB11 mice were pelleted at 500 g for 5 min at 4 °C, and whole cell pellets resuspended in 50μL ATAC-RSB buffer (10 mM Tris-HCl pH 7.4 10 mM NaCl; 3 mM MgCl2 , 0.1% Igepal CA-630), 0.1% Tween 20 (Sigma), and 0.01% Digitonin, and kept on ice for 3 min. 1ml of cold ATAC-RSB + 0.1% Tween20 was added, and samples centrifuged 500g for 10 min at 4°c (fixed angle rotor) to obtain nuclear pellet. Transposase reaction of open chromatin was achieved by resuspending free nuclei in tagmentation mix (22.5 μL Tagment DNA Buffer; 2.5 μL Tagment DNA enzyme; 25μL H2O) (Illumina, FC-121- 1030) and incubating at 37 °C for 30 min. Purification of DNA was performed with Diapure kit (Diagenode), according to the manufacturer’s protocol. Barcoding and amplification was performed using Nextera Index Kit (Illumina, FC-121-1011), as previously described56. Amplified ATAC-seq libraries were purified using Gene-Read Size Selection Kit (Qiagen, 180514) according to the manufacturer’s protocol. Quality and quantity of final ATAC-seq libraries were assessed with the High Sensitivity DNA kit (Agilent, 5067-4626) run on an Agilent 2100 Bioanalyzer. ATAC-seq libraries were sequenced using Illumina 125-bp paired-ends sequencing on a HiSeq2500 platform with, generating between 38 and 43 million reads per condition per biological replicate.