Ovarian tissues from PCOS and control mice were taken, and total RNA was extracted by the TRIzol method. RNA sequencing (RNA-seq) was conducted by Beijing Allwegene Technology Company Limited (Beijing, China). The cDNA library was constructed by polymerase chain reaction (PCR). RNA-seq was performed using the PE150 sequencing strategy of Illumina’s second-generation high-throughput sequencing platform. Poor quality RNA-seq reads and adapters were filtered out. Clean read data were aligned using Tophat2 and Cufflinks software to complete the transcriptome comparison (23 (link)).
Pe150 sequencing strategy
Manufactured by Illumina
The PE150 sequencing strategy is a laboratory equipment offered by Illumina. It enables paired-end sequencing with read lengths of up to 150 base pairs for each end of the DNA fragment.
Automatically generated - may contain errors
Lab products found in correlation
Second generation high throughput sequencing platform, by Illumina (4 mentions)
Bacterial genome dna extraction kit, by Tiangen Biotech (1 mentions)
High throughput sequencing platform, by Illumina (1 mentions)
Agilent 2100 bioanalyzer instrument, by Agilent Technologies (1 mentions)
Ultrapure rna kit, by CWBIO (1 mentions)
9 protocols using pe150 sequencing strategy
1
RNA-seq Analysis of PCOS Mouse Ovaries
2
Genomic Analysis of Carbapenem-Resistant CPKP
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Whole-genome sequencing was performed on 133 CPKP strains that completed the preliminary screening and phenotypic detection of carbapenemase. The whole-genome DNA of bacteria was extracted by Tiangen Bacterial Genome DNA Extraction Kit (Tiangen, Beijing, China). Sequencing libraries were prepared using NextEra Ultra. Two-terminal sequencing was performed on an Illumina II High-throughput Sequencing Platform and PE150 Sequencing Strategy. The whole-genome sequencing was commissioned by Shenzhen Haiyi Times Genomics. The genome of sequencing data was assembled by SOAPdenovo, and then the inner hole of the assembly result was repaired by GapCloser software. GeneMarks software was used to predict coding genes. The carbapenemase resistance genes were searched through the CARD database and ResFinder in CGE, and the annotations of the functional database were compared by BLAST software. Carbapenem resistance genes were screened out according to the annotation results of WGS.
3
Phillygenin's semi-inhibition curve exploration
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In the exploration of the semi-inhibition curve of phillygenin, the concentration of the bacterial suspension was adjusted to 1 × 108 CFU/ml, with drug action lasting (16, 32, 64, and 128 μg/ml) for 0, 2, and 8 h. Thereafter, the OD value at 600 nm (HITACHI, Japan) was measured with the semi-inhibitory drug concentration confirmed when the OD value remained unchanged. The bacteria were extracted for a transcriptome sequencing, which was completed by Nanjing Fengzi Bio-pharm Technology using the second-generation Illumina high-throughput sequencing platform and PE150 sequencing strategy. As a result, a total of 80,448,750 raw read pairs were measured with 79,746,845 clean read pairs obtained after quality control. Bowtie2 and Rockhopper were employed to carry out the comparison and sliced transcript analysis. Thereafter, all genes were quantitatively analyzed, and a total of 1,214 differential genes were identified. A functional enrichment analysis was performed on these differential genes to explore their features.
4
RNA-seq analysis of intestinal I/R and Caco-2 H/R
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For RNA-seq, total RNA was collected from I/R intestinal tissues as well as H/R Caco-2 cells using si-NC or si-TMEM2. cDNA libraries were constructed from total RNA to analyze gene expression differences. The concentration and purity of total RNA and the quality of the library were determined by Bosite Biotech (Shenyang, China). The PE150 sequencing strategy on the Illumina platform was used to sequence suitable libraries, and then the R package was used for subsequent data analysis.
5
RNA-seq Transcriptome Analysis Protocol
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Total RNA was extracted using the TRIzol method. RNA sequencing was completed by Allwegene Technology Inc in Beijing. The cDNA library was then constructed using PCR amplification. RNA‐seq was performed with the PE150 sequencing strategy by the Illumina second‐generation high‐throughput sequencing platform. RNA‐seq reads with inferior quality or adapters were filtered. Clean reads data were processed using Tophat2 and Cufflinks software to complete the alignment of transcriptomes. Genes not expressed in any sample were excluded from further analysis. Differentially expressed genes and transcripts were then filtered for false discovery rate (FDR)‐adjusted P‐values less than or equal to .05. RNA‐seq data have been deposited (PRJNA637758).
6
Differential Gene Expression in Roquin2-Expressing Cancer Cells
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MDA-MB-468/Roquin2, MCF7/Roquin2, A549/Roquin2, SMMC-7721/Roquin2 cell and their control cell (expressing GFP) were cultured for 36 h, and total RNA was extracted using Trizol method. The integrity of the cleaned RNA was confirmed by NanoDrop spectrophotometry. RNA sequencing was completed by the Allwegene Technology Inc. in Beijing. The cDNA library was then constructed using PCR amplification. RNA-seq was performed with the PE150 sequencing strategy by the Illumina second-generation high-throughput sequencing platform. RNA-seq reads with inferior quality or Adapters were filtered. Clean reads data were processed using Tophat2 and Cufflinks software to complete the alignment of transcriptomes. Genes not expressed in any sample were excluded from further analysis. Differentially expressed genes and transcripts were then filtered for FDR adjusted p-values less than or equal to 0.05. RNA-seq data were deposited in NCBI SRA (https://www.ncbi.nlm.nih.gov/sra ) (PRJNA668641).
7
Comprehensive RNA-seq Analysis of Inflamed Tissues
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RNA-sequencing (RNA-seq) analysis was performed by Allwegene Technology Inc, Beijing, China. Total RNA was extracted from the inflamed tissue samples of the control group, CFA group, and EA group, and its concentration, purity, and integrity were determined. The length of the RNA fragment was detected using the Agilent 2100 bioanalyzer instrument (Agilent). Then, a cDNA library is constructed by PCR amplification. After quality control, clean read pairs were obtained using the Illumina second-generation high-throughput sequencing platform with the PE150 sequencing strategy. Complete comparison and transcript splicing analysis was performed using star and Cufflinks software and then quantitative analysis on all genes. Gene expression levels were measured using the HTSeq software and quantified as the ratio of reads mapped to a gene to the gene length in kbp and expressed as the fragments per kb of transcript per million fragments mapped (FPKM).
8
Glomerular Tissue RNA Extraction and Sequencing
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Thirty mg glomerular tissue was used to isolate total RNA with the Ultrapure RNA Kit (CWBIO, CW0581M). Briefly, after trituration in 1 mL TRIzol, the homogenized tissue was added to chloroform for incubation for 5 min and shaken vigorously. After centrifugation, the upper water phase was moved into an adsorption column and then eluted with RNase‐free water. PCR amplification was performed to construct a cDNA library. RNA‐seq was done with the PE150 sequencing strategy on the Illumina second‐generation high‐throughput sequencing platform. Poor quality reads were filtered, while clean reads data were processed by Tophat2 and Cufflinks software to complete transcriptome comparison and fragment splicing analysis.
9
Transcriptomic Analysis of Differentially Expressed Genes
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Total RNA extraction and mRNA were enriched using magnetic beads with Oligo(dT) for cDNA library construction at Beijing Allwe Gene Technology Co., Ltd. (Beijing, China). Then, sequencing was performed by Illumina second-generation high-throughput sequencing platform using the PE150 sequencing strategy. The raw reads obtained from Illumina sequencing (FASTQ format) were then filtered to obtain clean reads. According to methods [20 (link)], DEseq (version 1.10.1) was used to identify the differentially expressed genes (DEGs) with | log2(fold change)| > 1 and p < 0.05, and that R (version 3.3.3) was then used for principal component analysis (PCA), volcano mapping, and clustering analysis. Meanwhile, the hypergeometric test was applied to perform significant enrichment analysis on pathways to identify those with significant enrichment of DEGs by the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway.
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