RNA-Seq Data Analysis Pipeline

mRNA was enriched from 1 µg total RNA using NEBNext^® Poly(A) mRNA Magnetic Isolation Module (New England Biolabs E7490L). The library was prepared from the mRNA enriched fraction using NEBNext^® Ultra™ II Directional RNA Library Prep (New England Biolabs E7765L) according to the manufacturer’s instructions. The library was sequenced on the Illumina HiSeq 2500 platform, generating 150 bp end reads. The reads were trimmed for adapters using Trimmomatic v0.36^{61 (link)} and aligned to GRCh38 genome assembly using STAR aligner v2.6^{62 (link)} and gene-wise read coverage was quantified using Feature Counts from the Subread package v1.63^{63 (link)}. All downstream analysis was performed using R 3.5.1⁶⁴. Genes with TPM below 1 in every sample were discarded. DESeq2^{65 (link)} was used to identify differentially expressed genes. Gene ontology pathway analysis were done using DAVID v6.8^{66 (link),67 (link)}, Enrichr^{68 (link),69 (link)}, and Panther v15.0⁷⁰.

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Lee C.Q., Kerouanton B., Chothani S., Zhang S., Chen Y., Mantri C.K., Hock D.H., Lim R., Nadkarni R., Huynh V.T., Lim D., Chew W.L., Zhong F.L., Stroud D.A., Schafer S., Tergaonkar V., St John A.L., Rackham O.J, & Ho L. (2021). Coding and non-coding roles of MOCCI (C15ORF48) coordinate to regulate host inflammation and immunity. Nature Communications, 12, 2130.

Publication 2021

NEBNext® Poly(A) mRNA Magnetic Isolation Module NEBNext® Ultra™ II Directional RNA Library Prep HiSeq 2500

Genes Genome Isolation Library Mrna Poly a mrna

Corresponding Organization :

Other organizations : Institute of Medical Biology, Agency for Science, Technology and Research, Duke-NUS Medical School, Institute of Molecular and Cell Biology, University of Melbourne, Genome Institute of Singapore, Nanyang Technological University

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Variable analysis

independent variables

MRNA enrichment using NEBNext® Poly(A) mRNA Magnetic Isolation Module
Library preparation using NEBNext® Ultra™ II Directional RNA Library Prep

dependent variables

Gene-wise read coverage quantified using Feature Counts
Differentially expressed genes identified using DESeq2
Gene ontology pathway analysis using DAVID, Enrichr, and Panther

control variables

Total RNA input of 1 µg
Sequencing on Illumina HiSeq 2500 platform, generating 150 bp end reads
Reads trimmed for adapters using Trimmomatic v0.36
Reads aligned to GRCh38 genome assembly using STAR aligner v2.6
Downstream analysis performed using R 3.5.1
Genes with TPM below 1 in every sample discarded

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