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Nanopore RNAseq profiling of female wildtype or Malat1-/- naïve CD4+ T cells and in vitro differentiated Th2 cells for 2 days.

ABSTRACT: Transcriptomic profiling of Malat1-deficient and WT CD4+ T cells at Day 0 (naïve CD4+ T cells) and Day 2 post-induction of differentiation under Th2 conditions. Naive CD4+ T cells isolated from spleens and lymphnodes of female mice. For Th2 polarisation, purified naïve CD4+ T cells (500,000 cells per well) were stimulated with 10 μg/ml plate-bound anti-CD3ε (clone 145-2C11) and 4 μg/ml soluble anti-CD28 (37.51) in RPMI 1640 in flat-bottom 96-well plates in the presence of 25 ng/ml mouse rIL-4 and 5 μg/ml anti–IFN-γ (XMG1.2).

ORGANISM(S): Mus musculus

PROVIDER: GSE278413 | GEO | 2025/08/18

REPOSITORIES: GEO

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Project description:Several versions of SWI/SNF complexes, BAF and PBAF, have been described based on unique subunit composition. We find that T cell development in the thymus and lymphoid periphery is largely normal in the absence of the PBAF-specific component BAF180. However, BAF180-deficient Th2 cells express high levels of the immunoregulatory cytokine IL-10. BAF180 binds directly to regulatory elements in the Il-10 locus but is replaced by BAF250 BAF complexes in the absence of BAF180, resulting in increased histone acetylation and CBP recruitment to the IL-10 locus. These results demonstrate that BAF180 is a repressor of IL-10 transcription in Th2 cells and suggest that the differential recruitment of different SWI/SNF subtypes can have direct consequences on chromatin structure and gene transcription. mouse primary T helper lymphocytes, naïve and Th2 cells, resting and stimulated, comparison of wild-type and BAF180/Pbrm1 deficient cells RNA from T helper cells was compared in the presence (WT) and absence (KO) of the Pbrm1/BAF180 gene. Comparison was made in 2 T helper subsets (Naive and Th2), each under 2 condtions (resting and stimulated), in triplicate (A,B,C). Resting naïve CD4+ T cells (N) were purified to 95% purity from the spleens and lymph nodes of 4-6 week old Balb/c mice (WT, or BAF180 cKO) using CD4+CD62L+ T cell Purification Kit II per manufacturer’s instructions (Miltenyi). Stimulated Naive cells (S) were prepared from resting naive cells by stimulation for 24 hours on anti-CD3 (1ug/ml), anti-CD28 (2 ug/ml) coated plates at 1-2 x 106/ml. Resting Th2 cells (2U) were prepared from resting naive cells by plating onto anti-CD3 (1ug/ml), anti-CD28 (2 ug/ml) coated plates at 1-2 x 106/ml in the presence of 10ng/ml IL-4, 10ug/ml anti-IFN-gamma. IL-2 (100U/ml) was added 24 hours later. Cultures were expanded in IL-2 (100U/ml) three days after initial culture. Stimulated Th2 cells (2S) were prepared from resting Th2 cells, stimulated with PMA (50ng/ml) and Ionomycin (500ng/ml) for 1.5 hours. RNA was harvested from 3 replicates of the primary Th cells under each condition. RNA was isolated using RNAeasy Kit (Qiagen), Quality and quantity of the total RNA was checked with the Agilent 2100 bioanalyzer using RNA 6000 Nano chips. RNA was labeled using the standard Illumina protocol and Illumina TotalPrep RNA Amplification Kit (Ambion; Austin, TX, cat # IL1791) Biotin labeled cRNA was hybridized to Illumina’s Sentrix MouseRef-8,v2 Expression BeadChips.

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Nanopore RNAseq profiling of female wildtype or Malat1-/- naïve CD4+ T cells and in vitro differentiated Th2 cells for 2 days.

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