Oocytes and GCs were collected from 4-wk-old wildtype and Erβnull rats with or without gonadotropin treatment. Total RNA was extracted using TRI Reagent (Millipore-Sigma) following the manufactures instructions. Approximately 500ng of total RNA was used for the RNA-seq library preparation. Libraries were prepared by using a TruSeq standard mRNA kit (Illumina) following manufacturer’s instructions. The cDNA libraries were sequenced at the Molecular Biology Core Laboratory of Mayo Clinic (Rochester, MN). RNA-Seq data were analyzed using CLC Genomics Workbench (Qiagen Bioinformatics). Gene expression values were reported as TPM (Transcripts per million base pairs). All the RNA seq data are available at SRA (SRX6376730, SRX6376729, SRX6376735, SRX6376752, SRX6376751, SRX6376757, SX6761695, SRX6761696, SRX6761697, SRX6761698, SRX6761699 and SRX67617) and the data articles are published in Data in Brief journal (Chakravarthi et al., 2020 (link); Chakravarthi et al., 2019 ).
Truseq standard mrna kit
Manufactured by Illumina
Sourced in United States
The TruSeq standard mRNA kit is a laboratory equipment product that enables the preparation of mRNA samples for sequencing. It provides a standardized workflow for isolating and purifying mRNA from total RNA samples.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Tri reagent, by Merck Group (1 mentions)
Clc genomics workbench, by Qiagen (1 mentions)
Nextseq 500 instrument, by Illumina (1 mentions)
Clc genomics workbench v11, by Qiagen (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Rneasy plant mini kit, by Qiagen (1 mentions)
Hiseq 2500 v4 platform, by Illumina (1 mentions)
Rnaqueous micro total rna isolation kit, by Thermo Fisher Scientific (1 mentions)
Dynabeads mrna direct kit, by Thermo Fisher Scientific (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
3 protocols using truseq standard mrna kit
1
RNA-seq Analysis of Oocytes and Granulosa Cells
2
Transcriptome Analysis of Seedling Responses
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Total mRNA was extracted from seedlings using Trizol® reagent (Ambion) and an RNeasy Plant Mini Kit (Qiagen). RNA quality control and quantification were carried out using a Bioanalyzer 2100 system (Aligent). cDNA libraries were prepared with a TruSeq standard mRNA kit (Illumina) according to the manufacturer's instructions. cDNA sequencing was performed using a 500‐high output v2 sequencing kit and an Illumina Nextseq500 instrument. Bioinformatics analyses were conducted in QIAGEN CLC Genomics Workbench (v11.0.1). The heat‐map was generated by matching the log2 fold‐change value to a colour scale in EXCEL.
3
RNA-seq Analysis of Arabidopsis Flower Stages
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For each biological sample, 5-10 mg of ovules from stages 11-12 flowers were dissected and pooled together for total RNA extraction using an RNAqueous-Micro Total RNA Isolation Kit (Invitrogen) according to the manufacturer's instructions. The yields were estimated by electrophoretic and spectrophotometric analyses (NanoDrop 2000; ThermoFisher Scientific, Waltham, MA, USA). The RNA integrity number (RIN > 6.5, 28S/18S >1.0) was verified using an Agilent 2100 Bio-analyzer (Agilent Technologies Inc., Santa Clara, CA, USA). Messenger RNA (mRNA) was extracted using Dynabeads mRNA DIRECT Kit (Invitrogen), Illumina cDNA libraries were prepared using TruSeq Standard mRNA kit (Illumina, San Diego, CA, USA) for paired-end sequencing (125 bp) on one lane of an Illumina HiSeq2500 V4 platform. For each sample, a minimum of 5G clean data (Q30>83%) was collected. The sequence reads were mapped to the Arabidopsis genome using TopHat software. Low-quality reads were filtered with FASTX-Toolkit (version 0.013), and uniquely aligned reads were counted using htseq-count. Differentially expressed genes were analyzed using DESeq2, and were defined by a 1.5-fold expression difference and q-value <0.05.
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