N = 3 to 5 independent donors (each dot represents one donor). Total RNA was extracted and the expression of the different RFX mRNAs was measured by RT-qPCR. (B) Naive T lymphocytes were stimulated with plate-bound anti-CD3 and anti-CD28 antibodies for the indicated times. This experimental setup, with 2 data points (average of technical duplicates for each donor), precludes a statistical assessment of the differences observed. N = 2 independent donors (each dot represents one donor). Total RNA was extracted and the expression of RFX7 was measured by RT-qPCR. (A) Naive and memory T lymphocytes were stimulated with plate-bound anti-CD3 and anti-CD28 antibodies for 3 days. S9 Fig: RFX7 expression in human T lymphocytes. gRNA, guide RNA PDAP1, PDGFA-associated protein 1 RNP, ribonucleoprotein. Cell proliferation was measured 4 days after transfection by BrdU incorporation assay. Cells were transfected with either 1 (blue line) or 2 (yellow line) gRNAs at the same time. (D) Jurkat T cells were transfected with Cas-9 RNPs to delete either MYB (left) or PDAP1 (right). In each case, the red dashed square highlights an example of deleted or mutated clone that was used for further analyses. (C) Clones targeted in the MIR150 gene were screened for the presence of a deletion across the miR-150 sequence by PCR, using primers located externally to the 2 gRNAs.
In a second screening strategy (right), when digested by T7 endonuclease I, a PCR product of approximately 600 bp is digested into 2 segments of 200 and 400 bp if a mutation or small indel is present in the amplified region. Shown are 9 deleted clones that were used for downstream analyses, one clone (H8) that showed no deletion and was discarded and one control clone. In the first screening strategy (left), a PCR product of 0.78 kb is detectable upon deletion of the genomic region between the 2 sgRNAs indicated in blue in the schematic representation, while the nondeleted band can be detected in some PCR reactions at approximately 5.4 kb. (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-9–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 ΔBS4: N = 9 pooled clones, 2 experiments ΔBS2/3/4: N = 2 pooled clones). (C) Individual clones with the desired genomic modifications in the 3′ 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 ΔBS2/3/4. (B) Example of gDNA screening for single clones ΔBS4 (left) and ΔBS2/3/4 (right). (A) Schematic representation of the PDAP1 3′ 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 (ΔBS2/3/4 and ΔBS4, respectively). S2 Fig: Genomic deletion of 3 miR-150 binding sites in the PDAP1 3′ 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 ≥ 1 and ≤ −1 Log 10 p-value ≥ 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.