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Sample GSM7667402

Query DataSets for GSM7667402

Status

Public on Aug 03, 2023

Title

intramuscular adipocytes, si-CPT1B-2

Sample type

SRA

Source name

goat intramuscular preadipocyte

Organism

Capra hircus

Characteristics

cell type: goat intramuscular preadipocyte
genotype: Knockdown of CPT1B gene

Treatment protocol

The negative control groups was treated with negative control interfering RNA (NC) and the experimental groups treated with interfering RNA that silences CPT1B gene (si-CPT1B).

Growth protocol

Goat intramuscular precursor adipocytes were cultured using DMEM medium (containing 10% fetal bovine serum, 1% double antibody) at 37°C and 5% CO2.

Extracted molecule

total RNA

Extraction protocol

Total RNA was isolated and purified using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer's procedure. The RNA amount and purity of each sample was quantified using NanoDrop ND-1000 (NanoDrop, Wilmington, DE, USA). The RNA integrity was assessed by Bioanalyzer 2100 (Agilent, CA, USA) with RIN number >7.0, and confirmed by electrophoresis with denaturing agarose gel.
Poly (A) RNA is purified from 1μg total RNA using Dynabeads Oligo (dT)25-61005 (Thermo Fisher, CA, USA) using two rounds of purification. Then the poly(A) RNA was fragmented into small pieces using Magnesium RNA Fragmentation Module (NEB, cat.e6150, USA) under 94℃ 5-7min. Then the cleaved RNA fragments were reverse-transcribed to create the cDNA by SuperScript™ II Reverse Transcriptase (Invitrogen, cat. 1896649, USA), which were next used to synthesise U-labeled second-stranded DNAs with E. coli DNA polymerase I (NEB, cat.m0209, USA), RNase H (NEB, cat.m0297, USA) and dUTP Solution (Thermo Fisher, cat.R0133, USA）. An A-base is then added to the blunt ends of each strand, preparing them for ligation to the indexed adapters. Each adapter contains a T-base overhang for ligating the adapter to the A-tailed fragmented DNA. Single- or dual-index adapters are ligated to the fragments, and size selection was performed with AMPureXP beads. After the heat-labile UDG enzyme (NEB, cat.m0280, USA) treatment of the U-labeled second-stranded DNAs, the ligated products are amplified with PCR by the following conditions: initial denaturation at 95℃ for 3 min; 8 cycles of denaturation at 98℃ for 15 sec, annealing at 60℃ for 15 sec, and extension at 72℃ for 30 sec; and then final extension at 72℃ for 5 min. The average insert size for the final cDNA library was 300±50 bp.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Description

The sample-2 of knockdown CPT1B
si2

Data processing

fastp
Sequence readswere trimmed for adaptor sequnce/low-quality sequence using fastp
Trimmed sequnence reads wrere mapped to ARS1 using hisat2 samtools
The transcript was assembled using htseq-count.
Assembly: ARS1
Supplementary files format and content: tab-delimited text files include raw counts for each Sample
Supplementary files format and content: tab-delimited text files include RPKM values for each Sample

Submission date

Jul 28, 2023

Last update date

Aug 03, 2023

Contact name

Yinggui Wang

E-mail(s)

YingguiWang326@outlook.com

Phone

18788705310

Organization name

Qinghai-Tibetan Plateau Animal Genetic Resource Reservation and Utilization Key Laboratory of Sichuan Province