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Transcriptome analysis of gene expression and single-nucleotide-resolution m6A sites in normal and mettl3 cKO testis samples (CLIP-Seq)

ABSTRACT: RNA was isolated from control and Mettl3 cKO mouse testes using the TRIzol (Invitrogen) reagent by following the manufactures's instructions. For all samples the RNA integrity was checked using an Agilent Bioanalyzer 2100. All samples showed a RIN (RNA integrity number) of higher than 9. For RNA-seq, approximately 2.5 µg of total RNA was then used for library preparation using a KAPA Stranded mRNA-Seq Kit Illumina® platform (KAPABIOSYSTEMS, KR0960) according to the manufacturer’s protocol.For m6A-miCLIP-seq, 6 ug of mRNA was first fragmented and immunoprecipitated with m6A antibody and thus obtained m6A containing RNA was subjected to cDNA library construction according to previously reported method [Linder et al. 2015; PMID: 26121403].All the libraries were sequenced using HiSeq3000 (Illumina) in paired-read mode, creating reads with a length of 101 bp. Sequencing chemistry v2 (Illumina) was used.

ORGANISM(S): Mus musculus

PROVIDER: GSE99772 | GEO | 2017/08/16

SECONDARY ACCESSION(S): PRJNA389580

REPOSITORIES: GEO

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Transcriptome analysis of gene expression and single-nucleotide-resolution m6A sites in normal and mettl3 cKO testis samples (RNA-Seq)

Project description:RNA was isolated from control and Mettl3 cKO mouse testes using the TRIzol (Invitrogen) reagent by following the manufactures's instructions. For all samples the RNA integrity was checked using an Agilent Bioanalyzer 2100. All samples showed a RIN (RNA integrity number) of higher than 9. For RNA-seq, approximately 2.5 µg of total RNA was then used for library preparation using a KAPA Stranded mRNA-Seq Kit Illumina® platform (KAPABIOSYSTEMS, KR0960) according to the manufacturer’s protocol.For m6A-miCLIP-seq, 6 ug of mRNA was first fragmented and immunoprecipitated with m6A antibody and thus obtained m6A containing RNA was subjected to cDNA library construction according to previously reported method [Linder et al. 2015; PMID: 26121403].All the libraries were sequenced using HiSeq3000 (Illumina) in paired-read mode, creating reads with a length of 101 bp. Sequencing chemistry v2 (Illumina) was used.

2017-08-16 | GSE99771 | GEO

m6A peaks in mouse brain sample.

Project description:RNA was isolated from 4 weeks old Prrc2a flox/flox whole brain using the TRIzol (Invitrogen) reagent by following the company manual. mRNA was isolated by using Dynabeads mRNA Purification Kit (Invitrogen) For all samples the RNA integrity was checked using an Agilent Bioanalyzer 2100. All samples showed a RIN (RNA integrity number) of higher than 9. Approximately 2.5 µg of total RNA was then used for library preparation using a TruSeq™ RNA Sample Prep Kit v2 (Illumina, San Diego, CA, USA) according to the manufacturer’s protocol.The libraries were sequenced using HiSeq3000 (Illumina) in paired-read mode, creating reads with a length of 101 bp. Sequencing chemistry v2 (Illumina) was used.

2019-05-16 | GSE100492 | GEO

Transcriptomics analysis of gene expression in control, Prrc2a conditional knockout, and Fto transgene mouse brain sample

Project description:RNA was isolated from hippocampus sample from 4 weeks old prrc2a flox/flox and prrc2a flox/flox;Olig2-cre or 4 weeks old wt and Fto Tg micemice using the TRIzol (Invitrogen) reagent by following the company manual. For all samples the RNA integrity was checked using an Agilent Bioanalyzer 2100. All samples showed a RIN (RNA integrity number) of higher than 9. Approximately 2.5 µg of total RNA was then used for library preparation using a TruSeq RNA Sample Prep Kit v2 (Illumina, San Diego, CA, USA) according to the manufacturer's protocol.The libraries were sequenced using HiSeq3000 (Illumina) in paired-read mode, creating reads with a length of 150 bp. Sequencing chemistry v2 (Illumina) was used.

2019-05-16 | GSE100491 | GEO

Network Modeling of Liver Metabolism to Predict Plasma Metabolite Changes During Short-Term Fasting in the Laboratory Rat: Liver Transcriptome Changes in Study 3

Project description:Purpose: The purpose of this study is to measure the changes in liver transcriptome in response to short-term fasting between 7 and 13 h where the rats were dosed with 2 ml/kg of saline vehicle at 0 h Methods: Total RNA was isolated from the liver, using TRIzol Reagent (Thermo Fisher Scientific, Waltham, MA) and the direct-zol RNA Mini Prep kit (Zymo Research, Irvine, CA). The isolated RNA samples were then submitted to the Vanderbilt University Medical Center VANTAGE Core (Nashville, TN) for RNA quality determination and sequencing. Total RNA quality was assessed using a 2100 Bioanalyzer (Agilent, Santa Clara, CA). At least 200 ng of DNase-treated total RNA with high RNA integrity was used to generate poly-A-enriched mRNA libraries, using KAPA Stranded mRNA sample kits with indexed adaptors (Roche, Indianapolis, IN). Library quality was assessed using the 2100 Bioanalyzer (Agilent), and libraries were quantitated using KAPA library Quantification kits (Roche). Pooled libraries were subjected to 75-bp paired-end sequencing according to the manufacturer’s protocol (Illumina HiSeq3000, San Diego, CA). Results: No genes were were found to be differentially expressed with a false discovery rate less than 0.1 Conclusions: There were no significant changes in liver gene expression between 7 and 13 h of fasting

2019-03-19 | GSE123987 | GEO

Network Modeling of Liver Metabolism to Predict Plasma Metabolite Changes During Short-Term Fasting in the Laboratory Rat: Liver Transcriptome Changes in Study 1

Project description:Purpose: The purpose of this study is to measure the changes in liver transcriptome in response to short-term fasting between 5 and 10 h where the rats were dosed with 6 ml/kg of polyethylene glycol vehicle at 0 h Methods: Total RNA was isolated from the liver, using TRIzol Reagent (Thermo Fisher Scientific, Waltham, MA) and the direct-zol RNA Mini Prep kit (Zymo Research, Irvine, CA). The isolated RNA samples were then submitted to the Vanderbilt University Medical Center VANTAGE Core (Nashville, TN) for RNA quality determination and sequencing. Total RNA quality was assessed using a 2100 Bioanalyzer (Agilent, Santa Clara, CA). At least 200 ng of DNase-treated total RNA with high RNA integrity was used to generate poly-A-enriched mRNA libraries, using KAPA Stranded mRNA sample kits with indexed adaptors (Roche, Indianapolis, IN). Library quality was assessed using the 2100 Bioanalyzer (Agilent), and libraries were quantitated using KAPA library Quantification kits (Roche). Pooled libraries were subjected to 75-bp single-end sequencing according to the manufacturer’s protocol (Illumina HiSeq3000, San Diego, CA). Results: No genes were were found to be differentially expressed with a false discovery rate less than 0.1 Conclusions: There were no significant changes in liver gene expression between 5 and 10 h of fasting

2019-03-19 | GSE123935 | GEO

m6A peaks in vehicle/entacapone (ENT)-treated inguinal White Adipose Tissue (iWAT) of DIO mouse.

Project description:RNA was isolated from Vehicle/Entacapone(ENT) treated mouse inguinal White Adipose Tissue using the TRIzol (Invitrogen) reagent by following the company manual. mRNA was isolated by using Dynabeads mRNA Purification Kit (Invitrogen). For all samples the RNA integrity was checked using an Agilent Bioanalyzer 2100. Approximately 5 µg of mRNA was used for m6A-IP. The immunoprecipitated m6A modified mRNAs were then used for library preparation using a TruSeq™ RNA Sample Prep Kit v2 (Illumina, San Diego, CA, USA) according to the manufacturer’s protocol.The libraries were sequenced using HiSeq 2500(Illumina) in paired-read mode, creating reads with a length of 101 bp. Sequencing chemistry v2 (Illumina) was used.

2020-11-16 | GSE106996 | GEO

Transcriptomics analysis of gene expression in wildtype and FTO knockout mouse liver

Project description:Fto conditional knockout mutation was generated using phage-based Escherichia coli homologous recombination systems. Construct targeting the third exon of mouse Fto gene was introduced into ES cell by homologous recombination. The mice with homozygous targeted allele were crossed to Albumin-Cre for deletion the third exon of mouse Fto gene in liver. Total RNA was isolated from Liver Tissue using the TRIzol (Invitrogen) reagent by following the company manual. For all samples the RNA integrity was checked using an Agilent Bioanalyzer 2100. Approximately 2.5 µg of total RNA was then used for library preparation using a TruSeq™ RNA Sample Prep Kit v2 (Illumina, San Diego, CA, USA) according to the manufacturer’s protocol.The libraries were sequenced using HiSeq3000 (Illumina) in paired-read mode, creating reads with a length of 101 bp. Sequencing chemistry v2 (Illumina) was used.

2022-01-26 | GSE125785 | GEO

Transcriptomics of siCTRL, siNSUN2 and ALYREF-RIP HeLa cells, and multiple mouse tissues

Project description:RNA was isolated from siCTRL, siNSUN2 and ALYREF-RIP HeLa cells, and multiple mouse tissues using the TRIzol (Invitrogen) reagent by following the company manual. Approximately 2.5 µg of total RNA was then used for library preparation using a TruSeq™ RNA Sample Prep Kit v2 (Illumina, San Diego, CA, USA) according to the manufacturer’s protocol.The libraries were sequenced using HiSeq3000 (Illumina) or HiSeq2500 in paired-read mode, creating reads with a length of 101 or 125 bp. Sequencing chemistry v2 or v4 (Illumina) was used.

2017-04-19 | GSE93750 | GEO

RNA seq from peritoneum, peripheral blood, spleen and tumors from TC-1 grafted mice

Project description:Splenocytes, peripheral blood cells, peritoneal cells and tumor cells were washed with PBS and were counted. Cells were centrifuged at 400 g for 5 min and supernatant was removed. Cells pellet (< 3 x 106 cells) was resuspend in 350 µl of RLT buffer (Qiagen). RNA was extracted with RNeasy Mini kit (Qiagen) according to manufacturer’s protocol. mRNA library preparation was realized following manufacturer’s recommendations (KAPA mRNA HyperPrep ROCHE). Library purity/integrity were assessed using an Agilent 2200 Tapestation (Agilent Technologies, Waldbrunn, Germany). Final 7 samples pooled library prep were sequenced on Nextseq 500 ILLUMINA with MidOutPut cartridge (2x130Millions of 75 bases reads), corresponding to 2x18Millions of reads per sample after demultiplexing.

2020-07-09 | E-MTAB-9271 | biostudies-arrayexpress

Targeted resequencing of phenology genes in a diverse panel of barley varieties

Project description:We provide raw gene sequences of 174 flowering time regulatory genes and gene othologs across a large barley population (895 barley lines) selected from a collection of landrace, cultivated barley, and research varieties of diverse origin. This set represents the whole variety of cultivated barley lifeforms, namely two- and six-row genotypes with winter, spring, and facultative growth habits. We applied a target capture method based on in-solution hybridization using the myBaits® technology (Arbor Biosciences, Ann Arbour, MI, USA) which is based on in-solution biotinylated RNA probes. Baits were designed for flowering time regulatory genes and gene othologs, and used for production of 80mer capture oligonucleotides for hybridization. Genomic DNA was extracted from leaves of a single two-week old barley plant per variety using the cetyl-trimethyl-ammonium bromide (CTAB) method. Physical shearing of genomic DNA was performed with an average size of 275 bp. Library preparation was conducted with KAPA Hyper Prep Kit (KAPA Biosystems, Wilmington, MA). Hybridization of customised RNA baits with capture pools was performed at 65°C for 24 hours. Each pooled sequence capture library was sequenced on an Illumina HiSeq3000 instrument using three lanes to generate paired-end reads per sample. Genome sequencing was conducted at AgriBio, (Centre for AgriBioscience, Bundoora, VIC, Australia).

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