(B) Clones targeted in the PDAP1 gene were identified by PCR with or without T7 endonuclease I digestion. (A) Clones targeted in the MYB gene were screened for deletion mutants by using PCR primers located externally to the gRNAs. S6 Fig: Screening strategies for CRISPR/Cas-9вЂ“targeted primary T-cell clones. BS, binding sites gDNA, genomic DNA PDAP1, PDGFA-associated protein 1 RT-qPCR, reverse transcription quantitative PCR sgRNA, single-guide RNA UTR, untranslated region. Twenty-four hours after transfection, expression of PDAP1 was measured by RT-qPCR (Scrambled control clones: N = 8 pooled clones, 2 experiments О”BS4: N = 9 pooled clones, 2 experiments О”BS2/3/4: N = 2 pooled clones). (C) Individual clones with the desired genomic modifications in the 3вЂІ UTR of the PDAP1 gene (or control clones, transfected with a scrambled sgRNA sequence) were pooled and transfected with either a miR-150 mimic or control oligonucleotide. We could identify only 2 clones with a partial deletion of О”BS2/3/4. (B) Example of gDNA screening for single clones О”BS4 (left) and О”BS2/3/4 (right). (A) Schematic representation of the PDAP1 3вЂІ UTR with indicated the locations of the predicted miR-150 BS (black), the sgRNAs (red), and the primers used for gDNA screening (green arrows) of cells and clones lacking 3 or 1 single miR-150 site (О”BS2/3/4 and О”BS4, respectively). S2 Fig: Genomic deletion of 3 miR-150 binding sites in the PDAP1 3вЂІ UTR abrogates miR-150 regulation. FC, fold change miRNA, microRNA RT-qPCR, reverse transcription quantitative PCR. Expression of miR-150 compared to control samples was measured by RT-qPCR (left), and cell proliferation was measured by BrdU incorporation assay (right).

(D) Jurkat T cells were transduced with a lentiviral vector (LV) to force miR-150 expression. Data are shown as fold change compared to resting day 0 (d0) cells. Total RNA was extracted and the expression of the indicated miRNAs measured by RT-qPCR. (C) Naive, T CM and T EM cell subsets were isolated from peripheral blood and were either left resting or activated with plate-bound anti-CD3 and anti-CD28 antibodies for the indicated times. Differentially expressed miRNAs (Log 2 ratio в‰Ґ 1 and в‰¤ в€’1 Log 10 p-value в‰Ґ 1.3) are shown in red. (B) Volcano plot representations of the same data an in (A), to compare miRNA expression across subsets. Each dot represents an independent donor. Raw data were normalized to the top 25 most expressed miRNAs and considered as expressed if following thresholds applied: at least 1 sample with more than 125 normalized reads and no more than 1 sample containing less than 100 normalized reads. Moreover, 100 ng of total RNA were used for the analysis. (A) Naive, T CM, and T EM lymphocytes were freshly separated from the peripheral blood of 4 independent donors. S1 Fig: miRNA expression in primary human T lymphocytes.

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