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

Description

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).

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Libraries for RNA-Seq were prepared at the Finnish Functional Genomics Centre, using Illumina TruSeq Stranded mRNA Sample Preparation Guide (part # 15031047). The quality of the libraries was confirmed with Advanced Analytical Fragment Analyzer, and the concentrations of the libraries were quantified with Qubit® Fluorometric Quantitation (Life Technologies). Nine samples were pooled and sequenced on one lane of NovaSeq 6000 SP v1.5 Next-Generation Sequencing platform with 2x150bp read length. Sequencing was performed at the Finnish Functional Genomics Centre. FastQC (v.0.11.8) (Andrews, 2010) was used to check the quality of the raw sequencing reads. Rsubread (v.2.6.4) (Liao et al., 2019) was used to align the reads to the human reference genome hg38 and count the read for each RefSeq gene. The data was normalized as CPM (counts per million) values using EdgeR (v.3.34.1) (Robinson et al., 2009). Log2 transformed normalized data was used for differential expression analysis using the ROTS tool (Suomi et al., 2017), as genes with the false discovery rate (FDR) ≤ 0.05. The cell pellet was lysed in a lysis buffer (4% SDS in 50 mM TRIS-HCl, pH 7.6) followed by Benzonase treatment for 10 minutes at room temperature. The extract was then clarified by centrifugation at 13,000 x g for 10 minutes. Protein concentration was determined using a DC assay. Subsequently, dithiothreitol (DTT) was added to a final concentration of 10 mM, followed by the addition of iodoacetamide (IAA) to a final concentration of 20 mM. After incubation for 30 minutes in the dark, the lysate was digested using the Suspension Trapping (STrap) method as described previously (Hailemariam et al., 2018). The dried peptides were reconstituted in a formic acid/acetonitrile mixture, and 800 ng of peptide mixture was analyzed using an EasynLC 1200 coupled to an Orbitrap Fusion™ Lumos™ mass spectrometer (Thermo Scientific). Protein identification and quantification was performed as described earlier (Andrabi et al., 2024). Overall, the proportion of missing (zero) values in the data was very low (0.22%). The MS2-level quantity data were obtained, and proteins quantified with only one peptide across the entire experiment were filtered out as unreliable. Subsequently, the data was offset with +1 and log2-transformed (related to Figure 3C-D). The DE proteins between the LINC01871-silenced and control T cells were identified using ROTS (Suomi et al., 2017), as proteins with FDR ≤ 0.05.

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