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Sample GSM4161073

Query DataSets for GSM4161073

Status

Public on May 22, 2020

Title

3595CA3

Sample type

SRA

Source name

CA3 region of the hippocampus

Organism

Rattus norvegicus

Characteristics

strain: F344
tissue: CA3 region of the hippocampus
age: 5 months
Sex: male

Extracted molecule

polyA RNA

Extraction protocol

Rats were briefly anesthetized with isoflurane Patterson Veterinary, Greenly, CO and swiftly decapitated. The brains were then rapidly removed, and the hippocampi were dissected according to previously described methods Lein 2004; Zeier 2011. The hippocampal regions DG, CA1, and CA3 were separated, placed in the tubes, and flash-frozen in liquid nitrogen. All samples were stored in at -80°C until processed. RNA was isolated from subregions of a hippocampus from a single hemisphere of each animal. RNA Isolation was performed according to methods previously described Ianov 2016; Barter 2019. Briefly, RNA was isolated using the RNeasy Lipid Tissue Mini kit Qiagen, catalog number 74804. Total RNA was treated with DNase using the RNase-Free DNase Set Qiagen, catalog number 79254. Concentration of total RNA was assessed using the NanoDrop 2000 spectrophotometer Thermo Fisher catalog number ND-2000 and the RNA integrity number RIN was quantified using the Agilent 2200 Tapestation system with High Sensitivity RNA Screen Tape Agilent Technologies, Santa Clara, CA. Mean RNA integrity across all samples was 9.05 SEM ± 0.02 with values ranging from 8.2 to 9.3. Poly-A-selection for mRNA was performed using 250 ng of isolated total RNA in the Dynabeads mRNA DIRECT Micro kit Thermo Fisher, catalog number 61021. External RNA Controls Consortium ERCC RNA Spike-In Control Mixes were included in all isolated mRNA samples Thermo Fisher, catalog number 4456740.
Library preparation was performed using the Ion Total RNA-seq Kit v2 Thermo Fisher, catalog number 4475936. Ion Xpress barcodes Thermo Fisher, catalog number 4475485 were included with libraries for multiplex sequencing. In summary, isolated mRNA was fragmented with RNase III then ligated to the Ion Adapter Mix v2. RNA samples were then reverse transcribed. cDNA from each biological replicate was uniquely barcoded and amplified with 16 PCR cycles. Library concentration from each sample was quantified using the Qubit dsDNA HS Assay Thermo Fisher, catalog number Q32851. DNA fragment size distribution was subsequently evaluated using High Sensitivity D1000 ScreenTape in the 2200 Tapestation system Agilent Technologies, Santa Clara, CA. Templates were prepared using the Ion Chef system Thermo Fisher, catalog number 4484177 and sequencing was performed using the Ion Proton system Thermo Fisher, catalog number 4476610 or Ion GeneStudio S5 system Thermo Fisher, catalog number A38194.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Ion Torrent S5

Data processing

ERCC analysis was performed with the ERCC analysis plugin in the Torrent. Spiked samples had an R2 above 0.9.
Ion Torrent derived reads were aligned to the rattus norvegicus genome rn6 using STAR 2.5.3a in the in the Partek Flow servers Partek, Inc., St. Louis, MO.
Gene read counts were generated from Binary Alignment Map BAM files and annotated to the genome using Ensembl version 92; Partek, Inc..
Reads from FASTQ files produced by the Ion Torrent System were trimmed from both ends until all samples’ collective average Phred quality score was > 25. Reads shorter than 25 base pairs bps were also excluded.
Gene lists were filtered to exclude genes with an average count of 5 or less averaged across all samples included in a comparison.
Normalization and pairwise differential expression analysis were conducted using the DESeq2 package version 3.5 for comparisons by age, cognitive performance or hippocampal subregion. The threshold of significance was set at p<0.025 for differences by age.
Genome_build: Ensembl version 92; Partek, Inc..
Supplementary_files_format_and_content: Comma-separated values .csv files that contain the normalized counts for each sample

Submission date

Nov 14, 2019

Last update date

May 22, 2020

Contact name

Tom Foster

Organization name

University of Florida

Lab

Tom Foster

Street address

1149 Newell Drive