GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM5778129

Query DataSets for GSM5778129

Status

Public on Jan 12, 2022

Title

Peromuscus_maniculatus_x_polionotus_F1_hybrid_burrowing_rep3

Sample type

SRA

Source name

Whole brain

Organism

Peromyscus maniculatus x Peromyscus polionotus

Characteristics

tissue: Whole brain
condition: Burrowing

Treatment protocol

We measured burrowing behavior using large sand-filled (Scott Pharma, Marlborough, MA, USA) enclosures (1.2 x 1.5 x 1.1 m) set up as previously described (Weber et al., 2013). We first ran all experimental animals through two “pretest” trials in the enclosures to allow them to acclimate and to confirm individuals burrowed in a species- (or hybrid-) typical manner. Next, we randomly assigned animals to a test trial cohort, “burrowing” or “control”.

Growth protocol

All experimental animals were males and housed in standard polysulfone cages (19.7 x 30.5 cm and 16.5 cm high; Allentown, New Jersey, USA) in same sex and genotype groups, until testing adults at approximately 50-80 days of age. Housing cages contained enrichment as previously described (Lewarch and Hoekstra, 2018). We maintained animals at 22 ºC with a 16:8h light:dark cycle and provided them with standard rodent food and water ad libitum. All procedures were approved by the Harvard University Institutional Animal Care and Use Committee (protocol 27-09-3)

Extracted molecule

total RNA

Extraction protocol

To capture behavior-relevant gene expression, we focused on the brain. Therefore, at the conclusion of the test trial, we immediately euthanized animals using CO2 inhalation and rapidly dissected whole brains in chilled PBS, flash-froze the sample in liquid nitrogen, and stored it at -80 °C. Later, we homogenized tissues using a TissueLyser (Qiagen, Venlo, Netherlands) in Trizol (ThermoFisher Scientific, Waltham, MA, USA), and extracted total RNA using 5Prime Phase Lock Gel Tubes Heavy (Quantabio, Beverly, MA, USA), followed by clean-up with RNeasy columns (Qiagen, Venlo, Netherlands).
We prepared RNA-seq libraries with a TruSeq Stranded mRNA Library Prep Kit, following manufacturer’s instructions (Illumina, San Diego, CA, USA), and assessed library quality prior to sequencing using a TapeStation 2200 (Agilent Technologies, Santa Clara, CA, USA).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Description

BWxPO_F1_3

Data processing

To compare gene expression both across treatments and species, we first removed low-quality and adaptor sequences using SeqPrep (https://github.com/jstjohn/SeqPrep).
The P. maniculatus genome was masked at sites with SNPs using the perl script MaskReferencefromBED (https://github.com/TheFraserLab/ASEr) to avoid reference genome mapping bias. Reads from all F1 hybrid samples were aligned to this masked genome using STAR v 2.4.2a (Dobin et al., 2013) in 2-pass mode.
We determined ASE at the read-level using the script GetGeneASEbyReads in the ASEr package (https://github.com/TheFraserLab/ASEr).
To test for allele-specific expression differences, we first filtered ASE calls from GetGeneASEbyReads requiring at least 1 count per allele from all genes per sample.
After filtering we computed an ASE ratio for each gene by calculating the log2 ratio of P. maniculatus allelic counts compared to P. polionotus allelic counts. Positive values correspond to a P. maniculatus allelic bias while negative values reflect a P. polionotus bias.
Genome_build: pman 1.0
Supplementary_files_format_and_content: Matrix table with allele counts

Submission date

Jan 07, 2022

Last update date

Jan 13, 2022

Contact name

Ryan York

E-mail(s)

ryanayork@gmail.com

Phone

6505754507

Organization name

Stanford University