Genomics

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Transcriptome analysis of control and ERK3-depleted Human Colonic Primary Epithelial cells (HCPECs)


ABSTRACT: RNA sequencing analysis of control and ERK3 knockdown HCPECs was performed to understand the physiological relevance of ERK3 in maintaining basal as well as LPS-mediated innate immune responses in HCPECs. Results revealed that ERK3 is required for the regulation of several key cytokines and chemokines even at the steady state. In particular, we discovered that ERK3 is indespansable for the transcriptional regulation of CXCL8/IL-8, CXCL10, MCP-1/CCL2 and GCP-2/CXCL6. Concomitantly, a functional interpretation of the differentially expressed (DE) genes with topGO indicated an important role of ERK3 in regulation of genes involved in, among others, immune responses and leukocyte chemotaxis. Overall design: Three biological replicates of Control (siCo) and ERK3-depleted (siERK3) HCPECs were challenged with LPS for 24 h. After the stimulation cells were washed with cold PBS and lysed with Trizol (Cat# 15596018, Ambion) according to the manufacturer’s instructions. Total RNA was quantified by a Qubit 2.0 fluorometer (Invitrogen) and quality was assessed using Agilent’s bioanalyzer 2100 and a RNA 6000 Nano chip (Agilent). Samples with RNA integrity number (RIN) > 8 were further subjected for RNA library preparation. Barcoded cDNA libraries were prepared from 300 ng of total RNA using the NEBnext Poly(A) mRNA Magnetic Isolation Module and NEBNext Ultra RNA Library Prep Kit for Illumina (NEB) according to the provided instruction. Library quantity was assessed on a Qubit 2.0 using Invitrogen’s Qubit HS assay kit and library size was determined using Agilent’s Bioanalyzer 2100 and a HS DNA assay chip. Barcoded RNA-Seq libraries were onboard clustered using HiSeq Rapid SR Cluster Kit v2 using 8pM and 59 bps were sequenced on an Illumina HiSeq2500 using a HiSeq Rapid SBS kit v2.

INSTRUMENT(S): Illumina HiSeq 2500 (Homo sapiens)

ORGANISM(S): Homo Sapiens

SUBMITTER: Federico Marini  

PROVIDER: GSE136002 | GEO | 2020-04-27

REPOSITORIES: GEO

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