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Sample GSM5719326

Query DataSets for GSM5719326

Status

Public on Nov 09, 2022

Title

alveolar macrophage, FB1-1, LncRNA-seq

Sample type

SRA

Source name

FB1-1

Organism

Sus scrofa

Characteristics

cell type: alveolar macrophage
treatment: Fumonisin B1 (FB1)

Treatment protocol

The cell viability of porcine alveolar macrophages (3D4/21) cells treated with different concentrations (0, 10, 20, 30, 40, 50, and 60μg/mL) of FB1 at different culture time points (24, 48, and 72 h) was measured on a Tecan Infinit 200 microplate reader (Tecan) platform using the Cell CountingKit-8 (CCK-8) kit (Dojindo), and finally 50μg/mL FB1 was induced for 24 h as the optimal treatment concentration and action time to investigate the cytotoxicity of FB1 on 3D4/21 cells. Porcine alveolar macrophages were seeded in 6-well plates at a density of 5 × 105 cells/mL and cultured in a 5% CO2 incubator at 37℃ for 24 h. FB1 at a final concentration of 50 µg/mL was added to the culture medium of the experimental wells, and the same amount of enzyme-free water was added to the culture medium of the control wells.

Growth protocol

Porcine alveolar macrophages (3D4/21) were cultured in RPMI-1640 (Gibco) medium containing 10% fetal bovine serum (Gibco) and 1% antibiotics (penicillin (100 U/mL), streptomycin (0.1 mg/mL) (Beijing Solarbio Science & Technology Co., Ltd.) at 37℃ in a saturated humidity constant temperature incubator continuously supplied with 5% CO2.

Extracted molecule

total RNA

Extraction protocol

Total RNA was extracted from the experimental samples using the Trizol (Invitrogen, USA) kit according to the instructions, RNA purity and concentration were preliminarily detected using a NanoDrop2000 spectrophotometer (Thermo Scientific, MA, USA), and RNA integrity was accurately quantified using an Agilent 2100 (Agilent Technologies, CA, USA) bioanalyzer.
We remove the rRNAs from the total RNA of the sample, retain mRNAs and ncRNAs, reverse transcribe the obtained RNA, purify the cDNA fragment using QiaQuick PCR kit (Qiagen, Venlo, Holland), repair the end, add PolyA, add sequencing linker, degrade the product by UNG (Uracil-N-Glycosylase) enzyme and amplify the product by PCR, and sequence the library by Illumina HiSeqTM4000.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 4000

Description

RNA-Seq.FB1_1ln

Data processing

Data quality control was performed on ATAC-seq raw reads obtained from the sequencer before information analysis, and low-quality reads containing adapter reads, reads containing more than 10% unknown nucleotides (N), and low-quality reads containing more than 50% low quality (Q-value≤20) bases were removed to obtain high quality clean reads. The processed reads were aligned to the pig reference genome (release Sscrofa11.1) using Bowtie2 v2.2.8 with reads aligned to mitochondrial genome discarded, and reads that are uniquely aligned were used for subsequent analysis. The distribution map of insert fragments of each sample was drawn by ATACseqQC. DeepTools was used to visualize the read distribution flanking transcription start sites (TSSs). Peak calling was performed using MACS v2.1.2 with a threshold of q-value < 0.05. Only the common peaks among replicates (with overlap more than 50%) were retained for analysis. Peak annotation was performed using ChIPseeker v1.16.1.
We used fastp v0.18.0for quality control and data filtering of raw reads from rRNA-depleted library, to remove reads containing adapters, with a N-containing proportion greater than 10%, with all A bases, or with bases with Q-value≤20 accounting for more than 50% of the whole reads. The processed reads were aligned to the pig reference genome (release Sscrofa11.1) using HISAT2 v2.1.0. To examine the relationship among different samples, Principal component analysis (PCA) was performed using R package gmodels (http://www.rproject.org/). Differentially expressed genes were identified by DESeq2, with cut-off: FDR<0.05 and foldChange≥2. The raw data of small RNA library was filtered to remove low quality reads containing more than one low quality (Q-value≤20) base or containing unknown nucleotides (N) in the data, to filter out reads without 3 'adapters, to filter out reads containing 5' adapters, and to filter out reads containing poly A. All clean tags were searched to identify known porcine miRNAs (exist miRNAs) using the miRbase database (release 22).
Genome_build: Sscrofa11.1
Supplementary_files_format_and_content: Peak calling was performed using MACS v2.1.2. Peak annotation was performed using ChIPseeker v1.16.1
Supplementary_files_format_and_content: LncRNA_CodeGenesExpression_fpkm.csv
Supplementary_files_format_and_content: all_mirna_tpm.xlsx

Submission date

Dec 06, 2021

Last update date

Nov 09, 2022

Contact name

靳健

E-mail(s)

jiayao.jiang7@gmail.com

Phone

18860895953

Organization name

扬州大学