Transcriptome and proteome of LINC01871-deficient human CD4+ T cells

CD4+ T cells were isolated from human umbilical cord blood samples collected from Turku University Hospital from full-term normal delivery. Mononuclear cells were isolated using Ficoll density gradient centrifugation. CD4+ cells were then enriched using bead-based positive isolation (Dynal CD4 Positive Isolation Kit; Invitrogen, Cat. no. 11331D). LINC01871 was silenced using two LNAs targeting different regions of the gene (LNA1: 5´-TTCGGCCTTTGGTAGT-3´; LNA2: 5´-ACAGATCGTCCACGGC-3´) or non-targeting LNA (NT) (5´-AACACGTCTATACGC-3´). Cells were transfected with LNAs as described before (Andrabi et al., 2024). Briefly, 4 million cells, resuspended in 100 µl OptiMEM medium (Gibco by Life Technologies, Cat. no. 31985-047), were transfected with 300 pmol of LNA) using Amaxa nucleofector system (Nucleofector 2C / U-014 program) (Lonza). After nucleofection, cells were rested in RPMI medium, supplemented with pen/strep, 2 mM L-glutamine and 10% FCS, for 24 h at 37°C followed by their activation, as described above. LINC01871-deficient T cells were activated for 48 h, harvested and reactivated for 30 minutes, followed by RNA-seq and MS analyses.
Cells were activated using plate-bound α-CD3 (3.75 μg/ml; Beckman Coulter, Cat. no. IM1304) and soluble α-CD28 (1 μg/ml; Beckman Coulter, Cat. no. IM1376) in RPMI 1640 medium (Sigma-Aldrich) supplemented with L-glutamine (2 mM, Sigma-Aldrich), antibiotics (50 U/ml penicillin plus 50 μg/ml streptomycin; Sigma-Aldrich) and 10% FCS. All cultures were maintained at 37°C in a humidified atmosphere of 5% (v/v) CO2 incubator.
The RNA-seq and mass spectrometry (MS) data were of good quality. The principal component analysis (PCA) revealed low variability within biological replicates and high variability between the sample groups NT, LNA1 and LNA2 across the two main principal components (Fig. 2I). Hundreds of genes were DE upon LINC01871 silencing both in RNAseq and MS data (FDR<0.05), but the effect sizes were small (Table S2-3).