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Sample GSM7838179

Query DataSets for GSM7838179

Status

Public on Oct 20, 2023

Title

F1_F_15462_midbrain

Sample type

SRA

Source name

brain

Organism

Peromyscus maniculatus x Peromyscus polionotus

Characteristics

tissue: brain
brain subregion: midbrain
strain: F1 hybrid (P. maniculatus x P. polionotus)
Sex: F

Extracted molecule

total RNA

Extraction protocol

For the whole brain dataset, a cohort of 59–137-day-old virgin male P. maniculatus (n=5) and P. polionotus (n=6) animals were euthanized by CO2 inhalation 7–10 h before the start of the dark period. Whole brain samples were then rapidly dissected, flash-frozen, and stored at -70°C. We homogenized the samples in TRIzol Reagent (Invitrogen, 15596026) using a Bio-Gen PRO200 homogenizer (PRO Scientific, Oxford, USA), and extracted RNA using a Direct-zol™ RNA MiniPrep Plus kit (Zymo Research, R2070), including a DNase I treatment; For the brain subregion dataset, a cohort of 82–85-day-old virgin male and female P. maniculatus, P. polionotus, and their F1 hybrids were euthanized by CO2 inhalation followed by cervical dislocation 3–10 h before the onset of the dark period. One cohort was used for dissection of the cerebellum, hippocampus, and hindbrain samples. In these samples, after the brain was removed, a dorsal-to-ventral incision was made posterior to the cerebrum to release the hindbrain and cerebellum. The cerebellar peduncles were cut, and the hindbrain and cerebellum were placed in tubes and flash frozen in liquid nitrogen. The petri dish with the remaining brain was then placed on ice, and the two cerebral hemispheres were cut apart. For each hemisphere separately, the hippocampus was severed from the dorsal fornix and gently scooped out of the hemisphere with a brush in ventral-to-dorsal direction (Lu, Airey, and Williams 2001), and then flash frozen. For a second cohort of animals, we dissected the brains and then immediately submerged them into embedding compound for cryosectioning (Tissue-Tek O.C.T.) and froze them on dry ice. We sectioned brains on a coronal plane at 150 μm using a cryostat at -20°C (Leica CM3050 S, Leica Biosystems Inc., Buffalo Grove, IL) and stored the sections at -70°C until we dissected the medial prefrontal cortex, septum, striatum, hypothalamus, amygdala, thalamus, and midbrain with sample corers according to anatomical landmarks. The sample cores were immediately transferred into homogenization buffer. After subregion dissection, we homogenized samples for 10 seconds in Maxwell RSC Homogenization Buffer (Promega, Madison, USA) with 2% 1-thioglycerol using a Bio-Gen PRO200 homogenizer (PRO Scientific, Oxford, USA) and then extracted RNA using the Maxwell RSC simplyRNA Tissue Kit (Promega).
We prepared each whole brain sequencing library from 1 μg of total RNA using a PrepX PolyA mRNA Isolation Kit (Wafergen, 40047) on the Apollo 324 NGS Library Prep System (Takara Bio USA Holdings, Mountain View, CA). We added Illumina indices and amplified libraries with 12 cycles of PCR and cleaned them using PCRClean DX beads (Aline Biosciences, Woburn, MA); We prepared brain subregion RNA-Seq libraries using an mRNA HyperPrep kit (Kapa Biosystems, Wilmington, USA) automated on a BioMek FXp (Beckman Coulter, Brea, USA). Post-capture, we fragmented mRNA for 6 min at 94°C for a target insert size of 200-300 base pairs. We amplified dual-indexed adapter-ligated libraries in 11 PCR cycles.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Data processing

Using cutadapt v.2.3, we trimmed very low quality bases (quality score <10) and applied a minimum length cutoff of 25 bp.
We then mapped trimmed reads of P. maniculatus and P. polionotus individuals to their respective reference genome, Pman_2.1 (GCA_003704035.1) or Ppol_1.3.3 (GCA_003704135.2), and in-house annotations generated with Comparative Annotation Toolkit using the GENCODE v15 primary-assembly annotation of Mus musculus (GRCm38 mm10) as reference. We competitively mapped reads from F1 hybrids to a diploid reference genome created by concatenating the two species assemblies and annotations. We performed read mapping with STAR v.2.7.0e (Dobin et al. 2013) in a transcriptome-aware manner.
We estimated gene expression with RSEM v1.3.1 and retained only genes present in both species’ annotations. For F1 samples, allele counts (reads mapped to a diploid reference) were summed up.
Assembly: GCA_003704035.1; GCA_003704135.2
Supplementary files format and content: tab-delimited text file of expected gene counts (rows) for each sample (columns)

Submission date

Oct 12, 2023

Last update date

Oct 20, 2023

Contact name

Andreas F Kautt

Organization name

Harvard University

Street address

16 Divinity Ave

City

Cambridge