GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM7073789

Query DataSets for GSM7073789

Status

Public on Aug 08, 2023

Title

cow, pair-fed, rep3

Sample type

SRA

Source name

liver

Organism

Bos taurus

Characteristics

tissue: liver
treatment: pair-fed

Extracted molecule

polyA RNA

Extraction protocol

Total RNA was isolated from each sample using TRIzol reagent (Invitrogen, Carlsbad, CA), Phasemaker Tubes (Invitrogen), and RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions with minor modifications. Specifically, tissue samples were homogenized in TRIzol and lysates were transferred to Phasemaker Tubes with chloroform for 20 min rotation at 4°C. The aqueous phase was then transferred to the gDNA elimination column for 1 min rotation at room temperature. The eluted RNA was washed with cold 70% ethanol, transferred to the RNeasy mini spin column, and washed with buffer RPE and RW1 (RNeasy Mini Kit). Finally, RNAs were eluted in RNAse-free water and concentrations were determined by a NanoDrop 1000 spectrophotometer (Thermo Scientific, Waltham, MA).
RNA quality was evaluated on Bioanalyzer 2100 (Agilent) and only samples passed the QC were proceed with the downstream process. Next, mRNA was enriched from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers followed by the second strand cDNA synthesis. The library was ready after a series of subsequent steps, including end pair, A-tailing, adapter ligation, size selection, amplification, and purification. Finally, the library was checked with Qubit and real-time PCR for qualification and evaluated on a bioanalyzer for fragment size distribution. Quantified libraries were pooled and sequenced on Illumina platforms in paired-end mode (2x150bp).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Description

PF_919

Data processing

The adaptor removal and quality control of the raw sequencing reads were carried out using fastp (v0.23.2) . Reads with a percentage of low-quality base (quality score < 20) > 40% were removed. Reads with length < 30 bp or with too much Ns (> 5%) were also removed in this study. The cow reference genome (ARS-UCD 1.2) was downloaded from the UCSC database, and the alignment of clean reads were performed with STAR (v2.7.9a) allowing no more than 3 mismatches. The raw read counts for each gene were extracted using featureCounts (v2.0.3).
Assembly: ARS-UCD 1.2
Supplementary files format and content: tab-delimited text files include raw read count for each Sample

Submission date

Mar 01, 2023

Last update date

Aug 08, 2023

Contact name

Guangsheng Li

E-mail(s)

gl357@cornell.edu

Organization name

Cornell

Department

Animal Science