Transcriptomics analysis of gene expression in mouse lung after PM2.5 exposure
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE122492 | GEO | 2019/10/24
REPOSITORIES: GEO
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Project description:Total RNA was isolated from mouse lung harvested at different stages using TRIzol® Reagent (Ambion). mRNA was extracted with using Dynabeads® mRNA Purification Kit (Ambion) and subjected to TURBO™ DNase (Invitrogen) treatment at 37°C for 30 min and ethanol precipitation. Thus purified mRNA was used for RNA-BisSeq library construction. Around 200 ng mRNA premixed with the in vitro transcribed Dhfr mRNA at a ratio of 300:1 (Dhfr mRNA serves as methylation conversion control) was fragmented to ~100-nt fragments for 1 min at 90°C in 10× RNA Fragmentation Reagent (Ambion), then stopped by 10× RNA stop solution (Ambion), and precipitated with 100% ethanol. The RNA pellet was resuspended in 100 µl bisulfite solution (pH 5.1), which is a 100:1 mixture of 40% sodium bisulfite (Sigma) and 600 µM hydroquinone (Sigma) and subjected to heat incubation at 75°C for 4.5 h. The reaction mixture was desalted by passing through Nanosep with 3K Omega 500/pk columns (PALL Corporation) with centrifugation. After washed with nuclease-free water and centrifuged for five times, the RNA was finally disolved in 75 μl nuclease-free water and then desulfonated by incubation with an equal volume of 1 M Tris-HCl (pH 9.0) at 75 °C for 1 h. After ethanol precipitation, the RNA was resuspended in 11 µl of RNase-free water and subjected to library construction. Reverse transcription was carried out with superscript II Reverse Transcriptase (Invitrogen) and ACT random hexamers. The following procedures were performed with the KAPA Stranded mRNA-Seq Kit (KAPA) according to the manufacturer’s instructions.Libraries were sequenced using HiSeq2500 (Illumina) in paired-read mode, creating reads with a length of 125 bp.
2019-10-24 | GSE122491 | GEO
Project description:Total RNA was isolated using TRIzol® Reagent (Ambion). The RNA concentration and quality were determined with NanoDrop, Qubit and agarose gel analysis. The Dynabeads® mRNA Purification Kit (Ambion) was used to enrich the mRNAs, which were used as templates for RNA-Seq library construction. RNA-Seq libraries was directly constructed by using KAPA mRNA Stranded mRNA-Seq Kit (KAPA) according to the manufacturer’s instructions. The libraries were sequenced on the Illumina HiSeq X-Ten platform at Novogene (Tianjin, CA) with paired-end 150 bp read length. The different genes expression between the control and NSUN2 knockdown cell were detected.
2019-07-02 | GSE133621 | GEO
Project description:Total RNA was isolated from zebrafish embryos harvested at different stages using TRIzol® Reagent (Ambion). mRNA was extracted with using Dynabeads® mRNA Purification Kit (Ambion) and subjected to TURBO™ DNase (Invitrogen) treatment at 37°C for 30 min and ethanol precipitation. Thus purified mRNA was used for RNA-BisSeq library construction. Around 200 ng mRNA premixed with the in vitro transcribed Dhfr mRNA at a ratio of 300:1 (Dhfr mRNA serves as methylation conversion control) was fragmented to ~100-nt fragments for 1 min at 90°C in 10× RNA Fragmentation Reagent (Ambion), then stopped by 10× RNA stop solution (Ambion), and precipitated with 100% ethanol. The RNA pellet was resuspended in 100 µl bisulfite solution (pH 5.1), which is a 100:1 mixture of 40% sodium bisulfite (Sigma) and 600 µM hydroquinone (Sigma) and subjected to heat incubation at 75°C for 4.5 h. The reaction mixture was desalted by passing through Nanosep with 3K Omega 500/pk columns (PALL Corporation) with centrifugation. After washed with nuclease-free water and centrifuged for five times, the RNA was finally disolved in 75 μl nuclease-free water and then desulfonated by incubation with an equal volume of 1 M Tris-HCl (pH 9.0) at 75 °C for 1 h. After ethanol precipitation, the RNA was resuspended in 11 µl of RNase-free water and subjected to library construction. Reverse transcription was carried out with superscript II Reverse Transcriptase (Invitrogen) and ACT random hexamers. The following procedures were performed with the KAPA Stranded mRNA-Seq Kit (KAPA) according to the manufacturer’s instructions.Libraries were sequenced using HiSeq2500 (Illumina) in paired-read mode, creating reads with a length of 125 bp.
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2019-07-02 | GSE133622 | GEO
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2024-05-10 | GSE221872 | GEO
Project description:RNA-Seq procedure was used for analysis of expression in HEK293T cells. Cells were separately lysed with Trizol reagent (Invotrogen). Total RNA was extracted from cell lysed with Trizol reagent and PureLink RNA Micro Kit (Invitrogen) according to manufacture instruction. RNA quality was checked with BioAnalyser and RNA 6000 Nano Kit (Agilent). Poly(A)+ RNA was purified with Dynabeads® mRNA Purification Kit (Ambion). Illumina library was prepared from poly(A)+RNA with NEBNextNEBNext® Ultra™II RNA Library Prep Kit for Illumina® (NEB) according to manual. Sequencing was performed on HiSeq1500 with 50 bp read length. At least 10 millions of reads were generated for each sample.
2019-12-03 | GSE130262 | GEO
Project description:RNA-Seq procedure was used for analysis of expression in melanoma cells. Cells were separately lysed with Trizol reagent (Invitrogen). Total RNA was extracted from cell lysed with Trizol reagent and PureLink RNA Micro Kit (Invitrogen) according to manufacture instruction. RNA quality was checked with BioAnalyser and RNA 6000 Nano Kit (Agilent). Poly(A)+ RNA was purified with Dynabeads® mRNA Purification Kit (Ambion). Illumina library was prepared from poly(A)+RNA with NEBNextNEBNext® Ultra™II RNA Library Prep Kit for Illumina® (NEB) according to manual. Sequencing was performed on HiSeq1500 with 50 bp read length. At least 14.8 millions of reads were generated for each sample.
2024-10-08 | GSE221876 | GEO
Project description:RNA-Seq procedure was used for analysis of expression in HEK293T cells. Heat shock treatments was performed for 20 min at 43°C. Then heat shock treated cells were incubated for 6 h at 37°C and replicates were separately lysed with Trisol reagent. Total RNA was extracted from cell lysed with Trisol reagent and PureLink RNA Micro Kit (Invitrogen) according to manufacture instruction. RNA Quality was checked with BioAnalyser and RNA 6000 Nano Kit(Agilent). Poly(A)+ RNA was isolated with Dynabeads® mRNA Purification Kit (Ambion). Illumina library was prepared from poly(A)+RNA with NEBNextNEBNext® Ultra™II RNA Library Prep Kit for Illumina® (NEB) according to manual. Sequencing was performed on HiSeq1500 with 50 bp read length.
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