ShRNA cloning, cell culture, transfections, and shRNA knockdown. Four shRNAs targeting the coding region or the 3′ untranslated region of all IKZF1 isoforms were designed using Block-iT RNAi Designer (https://rnaidesigner.thermofisher.com/rnaiexpress/). Single-stranded oligonucleotides for IKZF1 shRNA and a sequence with no corresponding target in the human genome were annealed and cloned into pENTR/H1/TO vector and used for electroporation of an IgG1-secreting human LCL, MATAT6. RNA was extracted to assess gene knockdown efficiency for each shRNA tested by quantitative PCR (qPCR), and the IKZF1 shRNA resulting in the most significant knockdown was used for subsequent experiments. More details can be found in the Supplementary Note.

RNA, complementary DNA, and real-time PCR. RNA was extracted from MATAT6 cells and stable shRNA lines, followed by on-column DNase (deoxyribonuclease) digestion and synthesis of complementary DNA and qPCR. qPCR gene expression assays were performed for IKZF1, FUT8, IKZF3, and HPRT1. Samples were run in triplicate. Relative gene expression level was determined using the comparative Ct (cycle threshold) method. Statistically significant differences in gene expression were determined using the paired t test. More details can be found in the Supplementary Note.

Western blot analysis. Whole-cell lysates were prepared by lysing cells in radioimmunoprecipitation assay buffer on ice. Jurkat nuclear extract and human embryonic kidney 293T lysates were used as positive and negative controls for IKZF1. Ten micrograms of total protein was reduced and denatured before separation on 4 to 12% Bis-Tris NuPAGE gel. Membranes were probed using rabbit polyclonal anti-Ikaros and mouse anti–α-tubulin antibodies. Secondary antibodies were horseradish peroxidase–conjugated anti-rabbit IgG or anti-mouse IgG. Antibody detection was achieved using the enhanced chemiluminescence detection system. More details can be found in the Supplementary Note.

Glycan profiling of secreted IgG. Five million cells from stable bulk cultures of shRNA-expressing MATAT6 cells were washed in phosphate-buffered saline and then resuspended in (serum-free) Opti-MEM. After 72 hours, conditioned media were collected by centrifugation and immediately frozen at −80°C. The glycan profile of secreted IgG in samples collected on at least two occasions from IKZF1 shRNA lines (n = 3) or random shRNA lines (n = 2) was determined by LCMS as described under the validation LCMS study in the Supplementary Note. A paired t test (GraphPad QuickCalcs) was performed to assess significant differences in glycosylation features.

Chromatin immunoprecipitation. ChIP assays were performed using ExactaChIP buffers (R&D Systems), as described by the manufacturer, except for the modifications described in the Supplementary Note. ChIP input was incubated with either anti-IKZF1 or goat IgG isotype control antibody overnight at 4°C with rotation. Five micrograms of biotinylated anti-goat IgG was added for a further 2 hours before the addition of streptavidin-agarose beads. Agarose beads were collected by centrifugation. After the final wash, Chelating resin solution was added to the beads, and the samples were boiled for 10 min. ChIP-PCR was performed using primers flanking a binding site upstream of FUT8 identified by ChIP-seq analysis in the GM12878 cell line to confirm that IKZF1-DNA complexes also occur in MATAT6 cells. PCR products were analyzed on 2% (w/v) agarose gel by electrophoresis in tris-borate EDTA buffer.

SMR/HEIDI analysis for pleiotropy with complex traits. To assess regional association concordance between IgG glycosylation and other complex traits (SMR/HEIDI test), we were able to download summary-level statistics including signed regression coefficient estimates and SE of these estimates for 10 traits (see the Supplementary Note for details). For each unique locus and glycan-trait combination for the test, we used only the SNP with the lowest P value in glycan GWAS. For all the available traits, the same procedure as outlined in the “SMR/HEIDI analysis for pleiotropy with gene expression” was applied.

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