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Sample GSM6434200

Query DataSets for GSM6434200

Status

Public on Jun 10, 2024

Title

neonatalSN, GF_96_66

Sample type

SRA

Source name

Sciatic nerves

Organism

Mus musculus

Characteristics

tissue: Sciatic nerves
strain: C57BL6/JZtm
condition: Germ free
developmental stage: Neonatal (P8-14)

Growth protocol

Neonatal (P8-14) and young adult (63-76 days old) germ-free (GF), gnotobiotic (colonized with Oligo-Mouse Microbiota12, OMM12) and SPF C57BL6/JZtm (complex microbiota) mice were obtained from the Central Animal Facility (Hannover Medical School, Hannover, Germany). Breeding and maintenance of GF and gnotobiotic mice was performed in plastic film isolators (Metall+Plastik GmbH, Radolfzell-Stahringen, Germany) located in a room with a controlled environment (20–22 °C, 50–55 % humidity) and 12-hour light/dark cycles. GF and gnotobiotic mice received pelleted 50 kGy gamma-irradiated feed (V1124-927, Ssniff Spezialdiäten GmbH, Germany) and autoclaved water ad libitum. SPF mice were housed in individually ventilated cages (XJ Edge, Allentown) in a room with a controlled environment (20–22 °C, 50–55 % humidity) and 12 h light/dark cycles. SPF mice received pelleted 50 kGy gamma-irradiated feed (1314 breeding diet for mice and rats, Altromin) and chlorinated water ad libitum. Routine microbiological monitoring according to FELASA recommendations (Mähler et al., 2014) and recommendations for maintaining gnotobiotic colonies (Nicklas et a., 2015) did not reveal any evidence of infection with common murine pathogens in SPF animals or contaminants in GF and gnotobiotic animals. In total, this study included tissue sampling from 42 adult and 41 neonatal animals. The animals were sacrificed by decapitation (adults ones after inducing anaesthesia in CO2 atmosphere). All procedures were performed in accordance with the German Animal Welfare Legislation and the principles of the Basel Declaration and recommendations of Directive 2010/63/EU, they were approved by the local Institutional Animal Care and Research Advisory Committee and registered with the animal care committee of Lower-Saxony, Germany.

Extracted molecule

total RNA

Extraction protocol

Neonatal and adult DRG and sciatic nerve total RNA was isolated using TRIzol reagent (Life Technology) and processed according to manufacturer's instructions.
Neonatal and adult DRG and sciatic nerve total RNA was isolated using TRIzol reagent (Life Technology) and processed according to manufacturer's instructions. Quality of the extracted RNAs was assessed using the RNA 6000 Nano Kit on Agilent 2100 Bioanalyzer (Agilent Technologies). Libraries were prepared using the TruSeq Stranded mRNA Sample Prep Kit (Illumina) following the manufacturer’s instructions. Sequencing of pooled dorsal root ganglia and peripheral nerves collected from young adult CGM, GF and OMM12 mice was performed on the Illumina NextSeq 500 platform. Sequencing of pooled dorsal root ganglia and peripheral nerves collected from neonatal CGM, GF and OMM12 mice was performed on the Illumina NextSeq 1000 platform.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

NextSeq 1000

Description

neonatalSN.norm_counts.cpm.tmm.txt.gz

Data processing

After quality controls with FastQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/), raw reads were aligned to the mouse reference genome (UCSC mm10/GRCm38) using STAR 2.7.1a (Dobin et al., 2013) (with parameters –outFilterMismatchNmax 999 –outFilterMismatchNoverLmax 0.04). Gene expression levels were quantified with featureCounts v1.6.3 (Liao et al., 2014) (options: -t exon -g gene_name) using GENCODE M23 annotation. Multi-mapped reads were excluded from quantification. Gene expression counts were next analysed using the edgeR package (Robinson et al., 2010). After low expressed genes filtration (1 count per million (CPM) in less than 3 samples), normalisation factors were calculated using the trimmed-mean of M-values (TMM) method (implemented in the calcNormFactors function).
Assembly: mm10/GRCm38
Supplementary files format and content: Tab-delimited text file of normalized read counts per gene/samples.

Submission date

Aug 05, 2022

Last update date

Jun 10, 2024

Contact name

Chiara Cicconetti

E-mail(s)

chiara.cicconetti@unito.it

Organization name

University of Turin

Department

Dept. of Life Sciences and Systems Biol