Computational protocol: Hippocampal Transcriptomic Profiles: Subfield Vulnerability to Age and Cognitive Impairment

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[…] Sequencing and analysis by using Ion Proton was performed at the University of Florida. Poly-A selection for the Ion Proton sequencer was performed with 250 ng of total RNA using the Dynabeads mRNA DIRECT Micro kit (Thermo Fisher, catalog number 61021) followed by library preparations with the Ion Total RNA-seq Kit v2 (Thermo Fisher, catalog number 4475936) with the addition of the Ion Xpress barcodes for multiplex sequencing (Thermo Fisher, catalog number 4475485). In brief, poly-A selection was performed by the base pairing of the poly-A tail of mRNA to the oligo (dT)25 sequence of the magnetic Dynabeads. Further, the mRNA was enzymatically fragmented by RNAse III, purified, ligated to the Ion adaptor mix, and reverse transcribed. The cDNA was uniquely barcoded per biological replicate and amplified with 16 cycles of PCR. The concentration of the libraries was quantified by the Qubit dsDNA HS Assay (Thermo Fisher, catalog number Q32851), and size distribution was evaluated with the High Sensitivity D1000 ScreenTape in the 2200 Tapestation system (Agilent Technologies).Template preparation was performed in the Ion Chef system and sequencing was completed in the Ion Proton (Thermo Fisher). Low quality reads were removed from the FASTQ files resulting in reads containing an average length of 134 bp. The Ion Proton data were aligned to the rattus norvegicus (rn5) genome using the two step alignment method with TopHat2 and Bowtie2 in the Partek Flow servers (Partek, Inc.). Aligned reads were summarized as gene-level counts (featureCounts 1.4.4). The FASTQ files from the Ion Proton have been submitted to NCBI's Gene Expression Omnibus (GEO) under the accession number: GSE97608. [...] Sequencing and analysis by using Illumina HiSeq was performed at the Translational Genomics Research Institute, Arizona. Sequencing libraries were prepared with 250 ng of total RNA using Illumina's Truseq RNA Sample Preparation Kit v2 (Illumina, Inc.) following the manufacturer's protocol. In brief, poly-A containing mRNA molecules were purified using poly-T oligo attached magnetic beads. The mRNA was then thermally fragmented and converted to double-stranded cDNA. The cDNA fragments were end-repaired, a single “A” nucleotide was incorporated, sequencing adapters were ligated, and fragments were enriched with 15 cycles of PCR. Final PCR-enriched fragments were validated on a 2200 TapeStation (Agilent Technologies) and quantitated via qPCR using Kapa's Library Quantification Kit (Kapa Biosystems) on the QuantStudio 6 Flex Instrument (ThermoFisher). The final library was sequenced by 50 bp paired-end sequencing on a HiSeq 2500 (Illumina). Illumina BCL files were converted and demultiplexed (bcl2fastq 2.17). FASTQ files were trimmed of adapter sequences (CutAdapt 1.8.3) and aligned to rn5 (STAR 2.5). Aligned reads were summarized as gene-level counts (featureCounts 1.4.4). Sequencing and quality control reports were generated (FastQC 0.11.4 and Qualimap 2.1.3). The FASTQ files from the Illumina platform have been separately submitted to GEO under the accession number: GSE101798. [...] For poly-A mRNA gene expression sequenced by Illumina and Ion Proton, pairwise differential expression analysis was conducted with the R package DESeq2 (1.10.1) to identify transcriptional changes due to age, cognitive performance, or subfield. For the transcriptional changes due to age and cognitive performance, a significant cut-off was set at p < 0.01. The resulting data sets for each region represent seed lists of differentially expressed genes. Validation of expression was performed using poly-A mRNA sequenced on the Ion Proton. For Ion Proton mRNA, a significance cut-off was set at p < 0.05 for one tailed-tests, with the tail specified by the direction of fold change (FC) determined by the Illumina seed list. Thus, the adjusted p-value for the combined tests was p < 0.0005. The following formula was used to calculate the false discovery rate (FDR) = (number of genes identified by Illumina * 0.0005/the number genes from this list identified by Ion Proton). Heat maps were generated in R with “gplots” (3.0.1) and “ComplexHeatmap” (1.14.0) using counts which were standardized to z-scores from genes validated with the Ion Proton, and the box plots were generated with “ggplot2” (2.2.1).To be labeled subfield-specific in CA1, CA3, or DG, gene counts had to be significantly different (Benjamini-Hochberg adj- p < 0.05) from both of the other two subfields with a concordant direction of fold change and validated by Ion Proton sequencing at the same significance threshold. Finally, pathway analysis was conducted with the ToppGene web portal against KEGG, PantherDB, and Reactome databases. An additional analysis was conducted using DAVID (version 6.8), considering Gene Ontology for biological processes and cellular components in the “Direct” and “FAT” categories with the Benjamini FDR set at p < 0.05 as a cut-off for cluster selection. […]

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