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Sample GSM7010005

Query DataSets for GSM7010005

Status

Public on Mar 01, 2023

Title

Ileum_133_NF

Sample type

SRA

Source name

Ileum

Organism

Mus musculus

Characteristics

tissue: Ileum
Sex: Male
strain: C57BL/6N
genotype: Neurog3f/f
diet: High fat diet

Growth protocol

RNA sequencing was carried out on the duodenum and ileum from adult Neurog3f/f; Villin-CreERT2 and control males (n=4/genotype) fed with a high fat diet, collected 5 weeks after tamoxifen administration.

Extracted molecule

total RNA

Extraction protocol

NucleoSpin RNA Plus, Mini Kit (Macherey-Nagel)
Total RNA-Seq libraries were generated from 500 ng of total RNA using TruSeq Stranded Total RNA Library Prep Gold kit and TruSeq RNA Single Indexes kits A and B (Illumina, San Diego, USA) following manufacturers' instructions except at the step of ribodepletion for which we used the riboPOOL kit targeting HMR ribosomal rRNA (siTOOLs Biotech, Planegg/Martinsried, DE) in replacement of the rRNA Removal Mix-Gold from Illumina. Following clean up on RNAClean xp beads, depleted RNAs were fragmented into small pieces using divalent cations at 94°C for 8 minutes. Cleaved RNA fragments were then copied into first strand cDNA using reverse transcriptase and random primers followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. Strand specificity was achieved by replacing dTTP with dUTP during second strand synthesis. The double stranded cDNA fragments were blunted using T4 DNA polymerase, Klenow DNA polymerase and T4 PNK. A single 'A' nucleotide was added to the 3' ends of the blunt DNA fragments using a Klenow fragment (3' to 5'exo minus) enzyme. The cDNA fragments were ligated to double stranded adapters using T4 DNA Ligase. The ligated products were enriched by PCR amplification (30 sec at 98°C; [10 sec at 98°C, 30 sec at 60°C, 30 sec at 72°C] x 12 cycles; 5 min at 72°C). Surplus PCR primers were further removed by purification using AMPure XP beads (Beckman-Coulter, Villepinte, France) and the final cDNA libraries were checked for quality and quantified using capillary electrophoresis.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

NextSeq 2000

Data processing

Image analysis and base calling were performed using RTA 2.7.7 and BCL Convert version 3.8.4.
Reads were preprocessed using cutadapt version 1.10 in order to remove adapter, polyA and low-quality sequences (Phred quality score below 20), reads shorter than 40 bases were discarded for further analysis.
Reads mapping to rRNA were discarded (this mapping was performed using bowtie version 2.2.8).
Reads were mapped onto the mm39 assembly of mouse genome using STAR version 2.5.3a.
Gene expression was quantified using htseq-count version 0.6.1p1 and gene annotations from Ensembl release 104.
Assembly: mm39
Supplementary files format and content: Tabulated text file containing raw read counts in each sample.

Submission date

Jan 30, 2023

Last update date

Mar 01, 2023

Contact name

Gérard Gradwohl

E-mail(s)

[email protected]

Organization name

IGBMC

Street address

1 rue Laurent Fries