Solid total rna seq kit

Frozen tissues ranging from 70–190 mg were homogenized in 2 ml TRIzol® reagent (Ambion) using an Ultratorrax T25 homogenizator (Labortechnik). Total RNA was extracted using RiboPure kit (Ambion) according to the manufacturers’ instructions. PolyA RNA was enriched from 1 μg total RNA using MicroPoly (A) Purist kit (Ambion) according to the manufacturer’s instructions. The quantity and quality of the input RNA was controlled using a RNA 6000 Pico chip on a Bioanalyzer (Agilent Technologies) and only RIN values above 7 were used in the analysis.
Complementary DNA (cDNA) library preparation was conducted at the Uppsala Genome Centre (SciLifeLab). Briefly, a ribosomal RNA (rRNA) depletion step was performed with 56 mg as input amount for all samples, using the RiboMinus Eukaryote kit (Life Technologies). Whole transcriptome libraries were then constructed using the SOLiD total RNA-Seq kit (rev B, July 2011, Life Technologies). Emulsion PCR was performed using the SOliD EZ Bead System (Life Technologies) and the libraries were then sequenced on three lanes with the SOLiD 5500xl System (Life Technologies).

Following the reproducible procedures of RNA extraction and linear amplification from our previous study [43 (link)], we isolated total RNA from individual oocytes/embryos using TRIzol (Invitrogen, Grand Island, NY) and co-precipitated the RNA with linear acrylamide (Ambion). The quality of the total RNA was examined with the Aglient RNA 6000 Pico kit (Aglient Technologies, Santa Clara, CA) using the Aglient Bioanalyzer 2100. RNA was then amplified twice using the TargetAmp 2-round aminoallyl-aRNA amplification kit 1.0 (Epicentre, Madison, WI) according to the manufacturer’s instructions. 500 ng of amplified RNA (aRNA) were used to construct the sequencing library following the manufacturer’s instructions by SOLiD™ Total RNA-seq Kit (Life Technologies, Grand Island, NY). After the sequencing library was prepared, we used an Agilent 2100 bioanlyzer to analyze the quality of the libraries. The sequencing libraries were then barcoded, multiplexed, and sequenced on a 5500xl Genetic Analyzer at the Center for Applied Genetics and Technology, University of Connecticut. We obtained 430 million sequencing reads with a read length of 75-bp from 16 single oocytes and embryos. The high correlation coefficients between samples of the same development stage demonstrated the reproducibility of the method (Additional file 2: Table S2).

mRNA-Seq library samples were prepared using a SOLiD Total RNA-Seq kit (Life Technologies). The amount of starting RNA material for RNA fragmentation was 500 ng. RNA samples were fragmented with RNase III, hybridized, and ligated to SOLiD adapters and ligation enzymes. cDNA was synthesized using ArrayScript Reverse Transcriptase and size-selected by separation on a TBE-urea gel. After amplification of cDNA, the yield and size distribution of the amplified cDNA were assessed using the Bioanalyzer. cDNA libraries were sequenced on SOLiD 3 plus (Life Technologies) with 50-bp single-end reads for whole-transcriptome sequencing (SRA accession number: SRX370383). Sequenced reads were uniquely aligned to the UCSC reference human genome (build hg19) using the whole-transcriptome mapping module in Bioscope 1.3.1 from Life Technologies, with default settings. BAM files were converted into BED files using BamTools.

Total RNA was isolated with Trizol in accordance with the manufacturer’s protocol (Life Technologies, Carlsbad, CA, USA) and treated with DNase I (Epicentre, Madison, WI, USA) to remove genomic DNA contamination. RNA purity was quantified using Qubit (Life Technologies). Seven micrograms of total RNA were used for ribosomal RNA (rRNA) depletion with MICROBExpress^TM kit (Thermo Fisher Scientific Inc., Idstein, Germany). The efficiency of depletion was evaluated in agarose gel electrophoresis (1%), followed by quantification of the total RNA with an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). A total of 500 ng mRNA was used for the construction of a sequencing library using the standard protocol of the SOLid Total RNAseq Kit (Life Technologies). The libraries were barcoded using the SOLiD Transcriptome Multiplexing Kit (Life Technologies). The emulsion PCR and sequencing were performed according to the Ion One Touch^TM 200 Template Kit v2 DL and the Ion PI^TM Sequencing 200 Kit v2 using the standard Life Technologies protocols, respectively. The Ion Proton Semiconductor Sequence (Life Technologies) was used to sequence 12 libraries generated from three biological replicates from each independent treatment.

Frozen tissue samples (70–190 mg) were homogenized in 2-ml TRIzol^® Reagent (Ambion) using a Ultra-Torrax T25 homogenizator (Labortechnik). Total RNA was extracted RiboPure Kit (Ambion) according to manufacturers´ instructions. Poly(A) RNA was enriched from 1-μg total RNA using MicroPoly(A)Purist Kit (Ambion) according to the manufacturer’s instructions. The quantity and quality of the input RNA were controlled using a RNA 6000 Pico chip on a Bioanalyzer (Agilent Technologies), and only RIN values above 7 were used in the analysis.
cDNA library preparation was conducted at the Uppsala Genome Centre (SciLifeLab). Briefly, an rRNA depletion step was performed with 56 mg as input amount for all samples, using the RiboMinus Eukaryote Kit (Life Technologies). Whole-transcriptome libraries were then constructed using the SOLiD Total RNA-Seq Kit (rev B, July 2011, Life Technologies). Emulsion PCR was performed using the SOLiD EZ Bead System (Life Technologies), and the libraries were then sequenced on three lanes with the SOLiD 5500xl System (Life Technologies).