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Sample GSM6329635

Query DataSets for GSM6329635

Status

Public on Jan 20, 2023

Title

WT SVE 2

Sample type

SRA

Source name

Suprachiasmatic nucleus

Organism

Mus musculus

Characteristics

strain: C57BL/6
genotype: wildtype
treatment: SVE

Treatment protocol

At the start of behavioral screening, mice were placed for 14 days in LD12:12 and then the lights were turned off and the animals maintained in constant dark (DD) for the remainder of the experiment. In DD, mice were either allowed to free-run for the duration of the experiment (nSVE cohort) or were included in a SVE cohort. For this SVE cohort, animals free ran for 14 days and then their exercise in the running-wheel was restricted for 6h every 24h. Entrainment to SVE was defined as when the onset of drinking activity showed ~24h periodicity for 6-7 successive days. Vipr2-/- animals rapidly respond to SVE and show entrainment within 6-9 days (Hughes et al., 2021), whereas Vipr2+/+, mice took 35-40 days. Mice that did not entrain to SVE were not used in subsequent gene expression assessment and similarly, Vipr2-/- mice that were arrhythmic in the nSVE condition were also excluded.

Growth protocol

All procedures were carried out under the UK Animals (Scientific Procedures) Act (1986) and approved by the University of Manchester Review Ethics Panel. Most experiments were conducted on adult male mice (>8 weeks of age) of two genotypes: C57BL/6 (wild-type, Vipr2+/+; Harlan, Blackthorn, UK) and transgenic mice lacking the VPAC2 receptor, Vipr2-/- (C57BL/6 background; initial breeding stock of Vipr2-/- donated by A. Harmar, University of Edinburgh). Mice were bred and maintained at 20–22 °C and ~40% humidity, with ad libitum access to food and water prior to and throughout experiments. Breeding rooms were maintained on a 12-h:12-h LD (LD 12:12) cycle.

Extracted molecule

total RNA

Extraction protocol

Once entrained, animals in the SVE cohort were culled 2h after the time at which the wheel became available to exercise in (ZT14). For animals in the nSVE cohort, they were culled at one timepoint (CT14). The brain was removed, flash frozen on dry ice, and stored at -80 C. Brain sections of 20µm thickness were cut using a cryostat (Leica CM3050 S) onto PEN-membrane slides (Leica Biosystems, Germany) and then stored at -80 °C. Slides were then defrosted and to aid in identifying the SCN and its anatomical boundaries, sections were stained with 1% cresyl violet (Merck) in 70% ethanol. The SCN was identified and extracted on a laser-capture microscope (LCM) system (Leica DM6000 B) and stored in lysis buffer (Promega) prior to RNA extraction. RNA extraction was carried out with ReliaPrep Kit (Promage, UK).
Total RNA was checked using RNA 6000 Pico Kit (Agilent Technologies, USA) and amplified with Sense Total RNA-Seq Library Prep Kit (Lexogen, USA). RNA sequencing was done by paired-end sequencing on an Illumina HiSeq 4000 NGS platform (Genomic Technologies Core Facility, University of Manchester, UK)

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 4000

Description

AH6

Data processing

Read trimming by Trimmomatic
QC with FastQC
aligment with HISAT2
Count and transcript assembly with Stringtie
Differential expression analysis with DESeq2
Assembly: GRCm39, vM27.annotations from GENCODE
Supplementary files format and content: DESeq2 output, tabular file containing normalised counts for all genes.

Submission date

Jul 12, 2022

Last update date

Jan 20, 2023

Contact name

Jean-Michel Fustin

E-mail(s)

Jean-Michel.Fustin@Manchester.ac.uk

Organization name

University of Manchester

Department

Centre for Biological Timing