Differentially regulated genes, P20 cerebral cortex, Slc13a4 heterozygous versus wild type mice
The rationale behind this profiling experiment was to understand the differential gene expression that arise during postnatal development in the mouse forebrain when one allele of Slc13a4 gene is absent. To do this, Slc13a4+/- mice and control wild-type littermates were used. These mice were maintained on a C57BL6 background. Wild-type females were bred with Slc13a4+/- males to generate Slc13a4+/- offspring. Polymerase chain reaction (PCR) confirmed the genotypes of each mouse. PCR primers are available on request. P20 Slc13a4+/- and control mice were cervically dislocated and whole brains were removed and placed on ice. The forebrain was isolated and snap frozen using dry ice. An RNeasy Micro Kit (QIAGEN) was used to extract total RNA from these samples, and 5–10 mg RNA in a total volume of 20 ml was sent to the Institute for Molecular Biosciences Sequencing Facility (The University of Queensland). The sequencing facility assessed sample quality using a Bioanalyzer. All samples passed the quality control with an RNA integrity number > 8. A second analysis was performed to measure the purity of the RNA using a spectrophotometer to determine the OD 260/280 ratio; all samples had values ~2.
RNA-Seq libraries were prepared using the Illumina TruSeq Stranded Total RNA LT (Ribo-Zero Gold) Sample Prep Kit (Illumina, RS-122-2301/RS-122-2302), according to the standard manufacturer’s protocol (Illumina, 15031048 Rev. E October 2013) described briefly as follows. To enrich for mRNA, 1 µg of total RNA was depleted of rRNA using Ribo-Zero Gold. The enriched mRNA was then subjected to a heat fragmentation step aimed at producing fragments between 130-290 base pairs (average 185 base pairs). cDNA was synthesised from the fragmented RNA using SuperScript II Reverse Transcriptase (Invitrogen, 18064014) and random primers. The resulting cDNA was converted into dsDNA in the presence of dUTP to prevent subsequent amplification of the second strand and thus maintaining the ‘strandedness’ of the library. Following 3’ adenylation and adaptor ligation, libraries were subjected to 15 cycles of PCR to produce libraries ready for sequencing. The libraries were quantified on the Perkin Elmer LabChip GX with the DNA High Sensitivity Reagent kit (Perkin Elmer, CLS760672). Libraries were pooled in equimolar ratios, and the pool was quantified by qPCR using the KAPA Library Quantification Kit - Illumina/Universal (KAPA Biosystems, KK4824) in combination with the Life Technologies Viia 7 real time PCR instrument.
Bulk RNA-sequencing was performed using the Illumina NextSeq500 (NextSeq control software v2.1.0 / Real Time Analysis v2.4.11). The library pool was diluted and denatured according to the standard NextSeq protocol (Document # 15048776 v02) and sequenced to generate paired-end 76 base pair reads using a 150 cycle NextSeq500/550 High Output reagent Kit v2 (Illumina, FC-404-2002). After sequencing, fastq files were generated using the bcl2fastq2 (v2.18.0) and received from the Institute for Molecular Bioscience Sequencing Facility (University of Queensland). Salmon software (v1.2.0) was used for quantifying transcript abundance. The count data was loaded into R using tximeta (Bioconductor v3.12). Differential gene expression analysis between Slc13a4+/- and wild-type samples was carried out in R using the DeSeq2 pipeline. Gene expression levels between Slc13a4+/- and wild-type samples were compared using Wald test as implemented in the DESeq2 pipeline. p-values were corrected using Benjamini-Hochberg adjustment. An adjusted p-value below 0.1 denoted a statistically significant difference in gene expression between samples. A fold change cut-off for differential expression of > ± 1.2 was also used to stratify differentially expressed genes.
