RNA-Seq Analysis Scripts to accompany: Balmas, E., et al (2018) Maternal group 2 innate lymphoid cells control fetal growth and protect from endotoxin-induced abortion in mice. doi:https://doi.org/10.1101/348755
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Balmas-Colucci-RNA-Seq.R
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README.md
tophat_doublemap.cfmod.pl

README.md

Maternal group 2 innate lymphoid cells control fetal growth and protect from endotoxin-induced abortion in mice

Elisa Balmas1,2,3,‡, Batika M. J. Rana 4,‡, Russell S. Hamilton2,3, Norman Shreeve1,3, Jens Kieckbusch1,3, Irving Aye1,3, Delia A. Hawkes1, Sophie Trotter1, Jorge López-Tello2,3, Hannah Yong2,3, Salvatore Valenti1, Amanda Sferruzi-Perri2,3, Francesca Gaccioli1,3, Andrew N. J. McKenzie4,‡ and Francesco Colucci1,3,‡,§

1 Department of Obstetrics and Gynaecology, University of Cambridge, University of Cambridge School of Clinical Medicine, NIHR Cambridge Biomedical Research Centre
2 Department of Physiology, Development and Neuroscience, University of Cambridge
3 Centre for Trophoblast Research, University of Cambridge
4 MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge CB2 0QH, UK
Equal contribution
§ Corresponding author: fc287@medschl.cam.ac.uk

Citation

Balmas, E., Rana, B.M.J., Hamilton, R.S., Shreeve, N., Kieckbusch, J., Aye, I., Hawkes, D.A., Trotter, S., López-Tello, J., Yong, H., Valenti., S., Sferruzi-Perri, A., Gaccioli, F., McKenzie, A.N.J. & Colucci, F. (2018) Maternal group 2 innate lymphoid cells control fetal growth and protect from endotoxin-induced abortion in mice. bioRxiv 348755

Abstract

Group 2 innate lymphoid cells (ILC2s) adapt to tissue physiology and contribute to immunity, inflammatory pathology and metabolism. We show that mouse uterine ILC2s have a heightened type-2 gene signature and expand during pregnancy. Indeed, maternal ILC2s protect against fetal growth restriction and mortality upon systemic endotoxin challenge. The absence of ILC2s leads to utero-placental abnormalities, including poor vascular remodelling, increased Il1b and decreased Il4, Il5, and Il13 gene expression, and reduced dendritic cells (DCs) and macrophage alternative activation. Placentas exhibit signs of adaptation to stress, including larger maternal blood spaces and increased expression of nutrient transporter genes. Endotoxin induces the expansion of IL-1-producing uterine DCs and, in response, more uterine ILC2s produce IL-4, IL-5 and IL-13. In a protective feedback mechanism, these cytokines suppress IL-1-producing uterine DCs, in line with a protective role of uILC2s against endotoxin-induced abortion. Uterine ILC2s emerge as pivotal for both normal and complicated pregnancies.

Data Processing

Data were aligned to GRCm38 mouse genome (Ensembl Release 86) with TopHat2 (v2.1.1, using bowtie2 v2.2.9) with a double map strategy. Alignments and QC were processed using custom ClusterFlow (v0.5dev) pipelines and assessed using MultiQC (0.9.dev0). Gene quantification was determined with HTSeq-Counts (v0.6.1p1). Additional quality control was performed with feature counts (v 1.5.0-p2), qualimap (v2.2) and preseq (v2.0.0). Differential gene expression was performed with DESeq2 package (v1.16.1, R v3.4.0) and with the same package read counts were normalised on the estimated size factors.

Resource URL
GRCm38 http://oct2016.archive.ensembl.org/index.html
FastQC http://www.bioinformatics.babraham.ac.uk/projects/fastqc/
Trim_galore http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/
TopHat2 https://genomebiology.biomedcentral.com/articles/10.1186/gb-2013-14-4-r36
ClusterFlow http://dx.doi.org/10.12688/f1000research.10335.2
MultiQC http://dx.doi.org/10.1093/bioinformatics/btw354
HTSeq-counts http://dx.doi.org/10.1093/bioinformatics/btu638
Feature_counts http://www.ncbi.nlm.nih.gov/pubmed/24227677
Qualimap https://doi.org/10.1093/bioinformatics/bts503
Preseq http://www.nature.com/nmeth/journal/vaop/ncurrent/full/nmeth.2375.html

A custom module for TopHat2 double map is provided in this repository, and can be run, by copying it into the modules directory of a ClusterFlow installation. With HTSeq-Counts gene count tables figures in the table below can be reproduced with the R script provided in this repository.

Script to reproduce paper figures

The provided R script assumes the script is placed in a directory containing a subdirectory (called HTSeq_Counts) with all the htseq-counts files (one per sample). The script can be run interactively in R-studio or as a batch using Rscript. Note that some of the figures in the manuscript have had some label positions moved manually to prevent overlaps.

Figure Description Output Filename
Figure 3A PCA 2018-Balmas-Colucci_DESeq2_Annotated_PCA_Fig3A.pdf
Figure 3B MA Plot 2018-Balmas-Colucci_DESeq2_MA_fc5_sig0.05_res_Uterine_Others_Fig3B.pdf
Figure 4A Heatmap 2018-Balmas-Colucci_DESeq2_CountMatrixHeatmap_topDEGs_lf7.5sig0.05_Fig4A.pdf
Figure 4B MA Plot 2018-Balmas-Colucci_DESeq2_MA_fc5_sig0.05_res_Virgin_E9.5_Fig4B.pdf
Figure 4D MA Plot 2018-Balmas-Colucci_DESeq2_MA_fc5_sig0.05_res_Virgin_E18.5_Fig4D.pdf

Additional Data

Description Output Filename
DEG Results: Uterine_vs_Others 2018-Balmas-Colucci_DESeq2_DEGs_Uterine_vs_Others.csv
DEG Results: Virgin_vs_E18.5 2018-Balmas-Colucci_DESeq2_DEGs_Virgin_vs_E18.5.csv
DEG Results: Virgin_vs_E9.5 2018-Balmas-Colucci_DESeq2_DEGs_Virgin_vs_E9.5.csv
Normalised reads counts 2018-Balmas-Colucci_DESeq2_NormalisedCounts.csv

Sample Table

sampleName condition cell tissue
SLX-10383.01 Virgin ILC2 Uterine
SLX-10383.02 Virgin ILC2 Uterine
SLX-10383.03 Virgin ILC2 Uterine
SLX-10383.04 E9.5 ILC2 Uterine
SLX-10383.05 E9.5 ILC2 Uterine
SLX-10383.06 E9.5 ILC2 Uterine
SLX-10383.07 E18.5 ILC2 Uterine
SLX-10383.08 E18.5 ILC2 Uterine
SLX-10383.09 E18.5 ILC2 Uterine
SLX-9189.04 Lymph_Node ILC2 Other
SLX-9189.05 Lung ILC2 Other
SLX-9189.06 Lymph_Node ILC2 Other
SLX-9189.07 Lung ILC2 Other
SLX-9189.08 Lymph_Node ILC2 Other
SLX-9190.01 Lung ILC2 Other

Links

Description URL
Publication bioRxiv
Raw Data ArrayExpress EMBL-EBI
Data to be released on publication
Uterine E-MTAB-5803
Lung/Lymph_Node E-MTAB-5806
Colucci Group Colucci group website

Contact

Contact rsh46 -at- cam.ac.uk for bioinformatics related queries