DOI: 10.5061/dryad.jm63xsjjc
title: MHC-II+ macrophage differentiation is impaired in metastasized lungs via PGE2 receptor EP2

description: Monocytes differentiate into macrophages (Mφs) to facilitate lung
 metastasis, but the monocyte-to-Mφ transition during this process is not
 well understood. To investigate, we performed bulk RNA sequencing on Mφs
 isolated from the lungs of mice bearing Lewis lung carcinoma tumors and
 from naive lungs. Our results showed impaired differentiation of monocytes
 into MHC-II+ Mφ, with upregulation of PGE2-inducible genes, including
 Arg1, in tumor-associated Mφs (TAMs). In vitro experiments confirmed that
 PGE2 inhibits the differentiation of MHC-II+ Mφ while promoting Arg1+ Mφ
 via the E prostanoid 2 (EP2) receptor, accompanied by DNA methylation.
 Whole genome bisulfite sequencing revealed that PGE2-EP2 signaling drives
 hypermethylation and downregulation of gene sets related to myeloid cells
 in non-neoplastic tissues. Our study highlights PGE2-EP2-driven DNA
 methylation in the monocyte-to-TAM transition, suggesting potential
 therapeutic avenues for lung metastasis.
description: <strong>NC and LLC Mφs RNA-seq</strong>
 mRNA-seq profiling of FACS-sorted lung macrophages
 (CD45+Ly6G-Ly6C-MerTK+CD64+SiglecF-CD11b+) from wild-type C57BL/6 male
 mice and Lewis Lung Carcinoma (LLC)-intravenously injected
 mice. RNA was harvested using Rneasy mini plus kit
 (Qiagen). 10ng of total RNA was used for the construction of sequencing
 libraries.  RNA libraries for RNA-seq were prepared
 using SMARTer Ultra Low RNA Kit (Takara) following manufacturer's
 protocols. <strong><em>In
 vitro </em>Control, PGE2-treated EP2 WT and EP2 KO BM cells
 RNA-Seq</strong> mRNA-seq profiling
 of GM-CSF-grown + IFNg+LPS 24 hours stimulated cells (Control),
 PGE2-treated EP2 WT and EP2 KO Bone marrow cells. RNA
 was harvested using Rneasy mini plus kit (Qiagen). 1ug of total RNA was
 used for the construction of sequencing libraries. 
 RNA libraries for RNA-seq were prepared using TruSeq Stranded
 mRNA Library Prep Kit (Illumina) following manufacturer's
 protocols. <strong><em>In
 vitro </em>Control, PGE2-treated EP2 WT and EP2 KO BM cells Whole
 genome bisulfite sequencing</strong> 
 WGBS profiling of GM-CSF-grown + IFNg+LPS 24 hours stimulated
 cells (Control), PGE2-treated EP2 WT and EP2 KO Bone marrow
 cells. DNA was harvested using QIAamp DNA Kits for
 DNA Extraction kit (Qiagen). 100ng of total RNA was used for the
 construction of sequencing libraries.  libraries for
 WGBS were prepared using Accel-NGS Methyl-Seq DNA Library Kit (Illumina)
 following manufacturer's protocols.
description: # MHC-II+ macrophage differentiation is impaired in metastasized lungs via
 PGE2 receptor EP2
 [https://doi.org/10.5061/dryad.jm63xsjjc](https://doi.org/10.5061/dryad.jm63xsjjc) **ALL of the RAW data were processed and prepared by Macrogen ([https://dna.macrogen.com/](https://dna.macrogen.com/))** **For macrophages sorted from NC (negative control/naive) and LLC lungs RNA-seq** * **Naive-IM1-3.fastq.gz / LLC-IM1-3.fastq.gz** * Samples were obtained by these procedures: * mRNA-seq profiling of FACS-sorted lung macrophages (CD45+Ly6G-Ly6C-MerTK+CD64+SiglecF-CD11b+) from wild-type C57BL/6 male mice and Lewis Lung Carcinoma (LLC)-intravenously injected mice. * RNA was harvested using Rneasy mini plus kit (Qiagen). 10ng of total RNA was used for the construction of sequencing libraries.  * RNA libraries for RNA-seq were prepared using SMARTer Ultra Low RNA Kit (Takara) following manufacturer's protocols. ***In vitro*** **Control, PGE2-treated EP2 WT and EP2 KO BM cells RNA-Seq** * **EP2_WT_PGE2-1-3.fastq.gz / EP2_KO_PGE2-1-3.fastq.gz / Control1-3.fastq.gz** * Samples were obtained by these procedures: * mRNA-seq profiling of GM-CSF-grown + IFNg+LPS 24 hours stimulated cells (Control), PGE2-treated EP2 WT and EP2 KO Bone marrow cells. * RNA was harvested using Rneasy mini plus kit (Qiagen). 1ug of total RNA was used for the construction of sequencing libraries.  * RNA libraries for RNA-seq were prepared using TruSeq Stranded mRNA Library Prep Kit (Illumina) following manufacturer's protocols. **All the RNA-seq RAW DATA (FASTq files) were processed through following steps:** * FastQC v0.11.7 was used to perform a quality check on data to ensure there are no issues before analyzing raw sequences. - Trimmomatic 0.38 program was used to perform trimming tasks on Illumina paired-end or single-end reads with various parameters. The parameters provided for the trimming step are as follows: * ILLUMINACLIP: Cuts adapters or specific sequences from the reads. * SLIDINGWINDOW: Performs sliding window trimming. If the average quality within the window is below a threshold, the read is trimmed. * LEADING: Trims bases from the start of the read if their quality is below a threshold. * TRAILING: Trims bases from the end of the read if their quality is below a threshold. * CROP: Trims the read to a specific length. * HEADCROP: Trims a specific number of bases from the start of the read. MINLEN: Drops reads that are shorter than a specified length. * TOPHRED33: Converts the quality score to a Phred33 score. * TOPHRED64: Converts the quality score to a Phred64 score. - HISAT2 version 2.1.0, Bowtie2 2.3.4.1 were used for a tool for fast and accurate mapping of high throughput sequencing data to a reference genome. It was the first to implement the graph FM index (GFM), based on the graph's BWT extension. Additionally, HISAT2 uses one global GFM and multiple small GFMs to create 55,000 indices representing 56 Kbp of the human genome, which allows for faster and more accurate sequencing read mapping. This type of indexing is referred to as the Hierarchical Graph FM index (HGFM). - StringTie version 2.1.3b was used for a tool that uses sequencing data mapped to a reference genome to quickly and accurately assemble potential transcripts. The novel network flow algorithm used in StringTie is applied not only in the de novo assembly stage but also for the quantification of multiple splice variants found in each gene region. ***In vitro*** **Control, PGE2-treated EP2 WT and EP2 KO BM cells Whole genome bisulfite sequencing (WGBS)** * **WGBS_Control1_1-2.fastq.gz / WGBS_KO_PGE2-1_1-2.fastq.gz / WGBS_WT_PGE2-1_1-2.fastq.gz** * Samples were obtained by these procedures: * WGBS profiling of GM-CSF-grown + IFNg+LPS 24 hours stimulated cells (Control), PGE2-treated EP2 WT and EP2 KO Bone marrow cells. * DNA was harvested using QIAamp DNA Kits for DNA Extraction kit (Qiagen). 100ng of total RNA was used for the construction of sequencing libraries.  * libraries for WGBS were prepared using Accel-NGS Methyl-Seq DNA Library Kit (Illumina) following manufacturer's protocols. * **RAW DATA (Fastq files) were processed through following steps:** * FASTQ File Example of FASTQ FASTQ file is composed of four lines. Line 1 : ID line includes information such as flow cell lane information. Line 2 : Sequences line. Line 3 : Separator line (+ mark). Line 4 : Quality values line about sequences. * FastQC v0.11.7 was used to perform a quality check on data to ensure there are no issues before analyzing raw sequences. - Trim Galore 0.5.0 was used for a wrapper script to automate quality and adapter trimming as well as quality control, with some added functionality to remove biased methylation positions for RRBS sequence files (for directional, non-directional (or paired-end) sequencing). - BSMAP 2.90 was used for a short reads mapping software for bisulfite sequencing reads. Bisulfite treatment converts unmethylated Cytosines into Uracils (sequenced as Thymine) and leave methylated Cytosines unchanged, hence provides a way to study DNA cytosine methylation at single nucleotide resolution. BSMAP aligns the Ts in the reads to both Cs and Ts in the reference. ## Description of the data and file structure All files are fastq