Sorted DN3a (15,000) (LinCD25+CD44CD27) and DN3b (LinCD25+CD44CD27+) cells were washed one time with cold PBS. Pellets were spin down at 500g for 5 min at 4°C, and the supernatant was removed carefully. Twenty microliters of transposase mix [10 μl of 2× TD (Tagment DNA) buffer, 1 μl of TDE (Tagment DNA Enzyme) (Nextera DNA Library Prep Kit; Illumina), 0.2 μl of digitonin (G9441, Promega), and 8.8 μl of nuclease-free water] was added to the cells. Reactions were incubated at 37°C for 30 min. Transposed DNA was purified using the MinElute Reaction Cleanup Kit (28204, Qiagen), amplified, and again purified according to published protocols (59). Size selection was performed using Low Range Ultra agarose (161-3107, Bio-Rad). Fragments between 150 and 600 bp in size were used for further analysis. Quality and quantity of the libraries were assessed by the Bioanalyzer High Sensitivity DNA Analysis Kit (Agilent) before sequencing. Libraries were sequenced 50 bp, paired-end, on HiSeq4000.

The reads were filtered by quality using TrimGalore tool (60) (default values), and the quality control was driven by FastQC (61) and MultiQC (62). The remaining reads were mapped to mm10 using bowtie2 (63) with –very-sensitive parameter. After all, before the peak calling, the read duplicates and multiple mapping reads were removed using Picard tools ( The peaks for two WT and two Tcf1−/− samples were called using MACS2 (64) with the following parameters: -g mm -B –shift -100 -ext 200 –nomodel –q 0.05 and BigWig-tracks with FPKM were generated by deepTools (65). Coverage plots and heat maps were generated with deepTools using the BigWig tracks previously generated with the following parameters: --binSize 100 -m 3000 -b 1000 -a 1000. To find differential open chromatin regions, the differential peaks between WT and Tcf1−/− conditions were calculated by DiffBind R/Bioconductor package (66), and only the statistically significant peaks (FDR < 0.05) were taken into account for downstream analysis. Motif analysis on the differentially accessible regions was performed using Homer ( using the parameters : size given. MEME-FIMO (67) and Tcf1 position probability matrix (MA07769.1) from JASPAR ( were used to analyze the distribution of the Tcf1 motif on the differentially accessible regions.

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