Transcriptomic changes associated with CCN2/CTGF deficiency in the mouse embryonic retina

The cellular communication network (CCN) 2, also known as CTGF, is a secreted matricellular protein with regulatory functions in vasoproliferative and fibrotic diseases. Although CCN2 functions extend to developmental processes, the impact of CCN2/CTGF expression, or lack thereof, during retinogenesis is unknown. Herein, we used bulk RNA-sequencing to determine the transcriptomic changes associated with global deletion of CCN2 in E18.5 mouse retinas. we show that CCN2 signals act on intrinsic signal transduction pathway genes and induce genetic reprogramming of retinal progenitor cells endowing these cells with neurogenic and gliogenic potentials. Retinal mRNA profiles of E18.5 wild-type (WTCCE) (obtained from mice with CCN2flox/flox genotype) and CCN2-deficient (KoCCE) mouse retinas (obtained from mice with CCN2flox/flox- CMV-Cre genotype) were generated by deep sequencing, in triplicate( WTCCE1, WTCCE2, WTCCE3 and KoCCE1, KoCCE3, KoCCE4)). Total RNA from each sample was enriched by oligo (dT) magnetic beads to remove rRNA. RNA-seq libraries were prepared using KAPA Stranded RNA-Seq Library Prep Kit (Illumina). The completed libraries were qualified with Agilent 2100 Bioanalyzer and quantified by absolute quantification qPCR method. To sequence the libraries on the Illumina HiSeq 4000 instrument, the barcoded libraries were mixed, denatured to single stranded DNA in NaOH, captured on Illumina flow cell, amplified in situ, and subsequently sequenced for 150 cycles for both ends on Illumina HiSeq instrument. Image analysis and base calling were performed using Solexa pipeline v1.8 (Off-Line Base Caller software, v1.8). Sequence quality was examined using the FastQC software. The trimmed reads (trimmed 5’, 3’-adaptor bases using cutadapt) were aligned to reference genome using Hisat2 software. The transcript abundance for each sample was estimated with StringTie, and the FPKM value for gene and transcript level was calculated with R package Ballgown. The differentially expressed genes and transcripts were filtered using R package Ballgown. The novel genes and transcripts were predicted from assembled results by comparing to the reference annotation using StringTie and Ballgown, then use CPAT to assess the coding potential of those sequences.