Project description:To identify the miRNAs expected to be potent in suppressing targets, we followed HMW RISC formation upon activation of CD8+ T cells. We show that while most miRNAs distribute between HMW and LMW RISC in activated T cells, several miRNAs were dominant in one complex over the other. Among these, miR-7 is enriched in HMW RISC and inhibition of miR-7 upon T cell activation leads to increased production of IL-2 and expression of IL-2-regulated proteins including the α-subunit of the IL-2 receptor, CD25; transferrin receptor, CD71; and amino acid transporter, CD98, which are direct miR-7 targets. Our data support a model where recruitment of miR-7 to HMW RISC restrains IL-2 signalling and the metabolic processes regulated by IL-2 and thus modulates T cell activation.
Project description:Splenic CD11b+ and CD4+ cells were isolated from Dnmt3a+/+ or Dnmt3aR878/+ mice using LS columns on the magnetic field of QuadroMACS Separator according to the manufacturer’s instructions (Miltenyi Biotec), and the High-Molecular-Weight (HMW) DNA of cells were isolated using MagAttract HMW DNA Kit according to the manufacturer’s instructions (Qiagen). WGBS was performed using the Zymo-Seq WGBS Library Kit (Zymo research) according to the manufacturer’s recommendations at the High Throughput Sequencing Core of Children’s Hospital of Philadelphia, PA, USA. The sequence was generated on Illumina NovaSeq 6000 using NovaSeq 6000 S4 Reagent Kit v1.5 (200 cycles) at 40X coverage
Project description:We used the nanopore Cas9 targeted sequencing (nCATS) strategy to specifically sequence 125 L1HS-containing loci in parallel and measure their DNA methylation levels using nanopore long-read sequencing. Each targeted locus is sequenced at high coverage (~45X) with unambiguously mapped reads spanning the entire L1 element, as well as its flanking sequences over several kilobases. The genome-wide profile of L1 methylation was also assessed by bs-ATLAS-seq in the same cell lines (E-MTAB-10895).
