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Sample GSM6760239

Query DataSets for GSM6760239

Status

Public on Jan 20, 2023

Title

Y25_3

Sample type

SRA

Source name

Liver

Organism

Mus musculus

Characteristics

tissue: Liver
Sex: Female
treatment: High-fat diet (HF)
time: Dark phase (Zeitgeber time 12-24)
time point: 20h
strain: C57BL/6

Extracted molecule

total RNA

Extraction protocol

Liver tissues were extracted via Qiazol method (RNeasy Tissue Lipid kit, Qiagen, MD USA). Concentration quantified via Spectrophometry 8000 (Thermofisher) and integrity on Qubit. Samples were sent off to Novogene USA for the complete analysis (mRNA methylation to total analysis of data).
Sequencing libraries were generated from 1 μg RNA per sample via NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations and index codes were added to attribute sequences to each sample. mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H.
Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3’ ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 150~200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptor ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. Clustering of the index-coded samples was performed on a cBot Cluster Generation System using PE Cluster Kit cBot-HS (Illumina) according to the manufacturer’s instructions. Library preparations were sequenced on an Illumina platform where paired-end reads were generated.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Description

Y25_3
mRNA

Data processing

Raw data (fastQ format) processed via fastp which clean data (clean reads) were obtained by removing reads containing adapter and pol-N- sequences. GC and phret scores were calculated at this time.
Clean paired-end reads were aligned to reference genome (obtained from NCBI) via STAR (version v2.5).
Quantification via HTSeq v0.6.1.
Differential Analysis via DESeq 2 (version v2_1.6.3, padju<0.05).
Enrichment Analysis via ClusterProfiler (version v2.4.3 with padj<0.05). Alternative splicing via rMATS (version v3.2.1 on default parameters) and Fusion gene analysis via STAR-Fusion (version v0.8.0 on default parameters).
Assembly: mm10
Supplementary files format and content: readcount_genename.xls: Raw counts of sequencing reads (before normalization) is provided in the matrix file. Column 1 provides the Ensembl ID, Column B-AQ provide the raw counts for each sample, columns AR-AY provide gene description (name, chromosomes, location, etc.)

Submission date

Nov 28, 2022

Last update date

Jan 20, 2023

Contact name

Lin Yan

E-mail(s)

[email protected]

Organization name

USDA- Grand Forks Human Nutrition Research Center

Street address

2420 2nd Ave N