Total RNA from bacterial cultures was extracted using the SV Total RNA Isolation System kit (Promega) following the manufacturer's instructions for Gram-negative Bacteria. Infected plant RNA extractions were carried out as described (Cruz et al., 2014 (link)). RNA concentration and quality was measured using the Agilent 2100 Bioanalyzer. For rRNA depletion, 2.5 μg of RNA were treated with the Ribo-zero(™) magnetic kit for bacteria (Epicenter). Three biological replicates per condition were subjected to sequencing on an Illumina-Solexa Genome Analyzer II apparatus in the Shanghai PSC Genomics facility using multiplexing and kits specially adapted to obtain 100 bp paired-end reads in stranded libraries. Raw sequencing data is available in the Sequence Read Archive under the accession code SRP096020 .
Solexa genome analyzer 2
Manufactured by Illumina
Sourced in United States
The Solexa Genome Analyzer II is a high-throughput sequencing system designed for DNA and RNA analysis. It utilizes a proprietary sequencing-by-synthesis technology to generate large volumes of sequencing data. The system is capable of producing gigabases of sequence data per run, enabling researchers to conduct a wide range of genomic studies.
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Lab products found in correlation
Trizol, by Thermo Fisher Scientific (2 mentions)
Ribo zero magnetic kit for bacteria, by Illumina (1 mentions)
Sv total rna isolation system kit, by Promega (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Rna 6000 nano kit, by Agilent Technologies (1 mentions)
Gel extraction mini elute kit, by Agilent Technologies (1 mentions)
Phusion dna polymerase, by Thermo Fisher Scientific (1 mentions)
Dna clean and concentrator 5 kit, by Zymo Research (1 mentions)
Ribo zero depletion, by Illumina (1 mentions)
Dynabeads oligo dt 25, by Thermo Fisher Scientific (1 mentions)
Model 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Sequencing adapter, by Illumina (1 mentions)
Hiseq 2500 platform, by Illumina (1 mentions)
Truseq stranded total rna sample preparation kit, by Illumina (1 mentions)
Nanodrop nd 1000 spectrometer, by Thermo Fisher Scientific (1 mentions)
Zymo dna clean and concentrator 5 kit, by Zymo Research (1 mentions)
5 protocols using solexa genome analyzer 2
1
Bacterial and Plant RNA Extraction and Sequencing
2
Total RNA Extraction and Small RNA Sequencing
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Total RNA was extracted using Trizol (Invitrogen) according to the manufacturer’s protocol, and the quantity and integrity of RNA were examined using a 2100 Bioanalyzer with the RNA 6000 Nano Kit (Agilent Technologies, Santa Clara, CA, USA). Sequencing libraries were generated using NEBNext® Multiplex sRNA Library Prep Set for Illumina® (NEB, USA.) following manufacturer’s recommendations. Each sample build a library, all the nine libraries were for sRNA sequencing using Illumina Solexa Genome Analyzer II was according to the manufacturer’s protocol (Illumina, San Diego, CA, USA).
3
ChIP-seq Library Preparation Protocol
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Single-end Solexa sequencing libraries were prepared as previously described (Schmidt et al, 2009 (link)). Briefly, 54 μl of ChIP DNA or 50 ng of total input control DNA were subjected to end repair using T4 DNA polymerase, Klenow DNA polymerase and T4 polynucleotide kinase, before purification using the DNA Clean and Concentrator-5 kit (Zymo Research). Adenine overhangs were added using Klenow 5′-3′ exo-minus, Illumina Solexa sequencing adapters were ligated using T4 DNA ligase and amplified with 18 PCR cycles using Phusion DNA polymerase (Finnzymes) and Illumina Solexa sequencing primers 1.1 and 2.1. Libraries were size selected by electrophoresis, excising the SYBR-safe, DNA smear between 200–300 bp on a Dark Reader non-UV transilluminator, purified using a Qiagen Gel Extraction MiniElute Kit, quantified using an Agilent Bioanalyser, 36 bp sequence reads were generated using a Illumina (Solexa) Genome Analyzer II and these reads were mapped back to the reference human genome before peak calling.
4
RNA Extraction and RNA-seq Library Preparation
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RNA was isolated from each human tissue sample by the use of TRIzol (Invitrogen, CA, USA) according to the instructions provided by the manufacturer. Total RNA quality and quantity were verified by the use of a model 2100 Bioanalyzer (Agilent Technologies, CA, USA) and a NanoDrop ND-1000 spectrometer (Thermo Scientific, DE, USA), respectively.
RNA-seq 1 was performed as described previously (42 (link), 43 (link)). Briefly, polyadenylated [poly(A)+] RNA was obtained with two rounds of poly(A) selection using Dynabeads Oligo(dT)25 (Invitrogen). DeLi-Seq was used to prepare the sequencing library and then purified by the use of a Zymo DNA Clean and Concentrator-5 kit (Irvine, CA). The products (∼200 to ∼300 bp) were sequenced using Illumina/Solexa Genome Analyzer II.
For RNA-seq 2, libraries were prepared using a TruSeq stranded total RNA sample preparation kit with Ribo-Zero depletion (Illumina, CA, USA). In brief, high quality total RNA (1 µg) was subjected to Ribo-Zero depletion, fragmented, and then subjected to reverse transcription (RT). Double-stranded cDNAs were A-tailed and ligated with Illumina sequencing adapters. Subsequently, the ligated products were enriched by PCR and size-selected by agarose gel electrophoresis. The products (∼200 to ∼400 bp) were sequenced by the use of an Illumina Hi-seq 2500 platform with a paired 50-mer sequencing modality.
RNA-seq 1 was performed as described previously (42 (link), 43 (link)). Briefly, polyadenylated [poly(A)+] RNA was obtained with two rounds of poly(A) selection using Dynabeads Oligo(dT)25 (Invitrogen). DeLi-Seq was used to prepare the sequencing library and then purified by the use of a Zymo DNA Clean and Concentrator-5 kit (Irvine, CA). The products (∼200 to ∼300 bp) were sequenced using Illumina/Solexa Genome Analyzer II.
For RNA-seq 2, libraries were prepared using a TruSeq stranded total RNA sample preparation kit with Ribo-Zero depletion (Illumina, CA, USA). In brief, high quality total RNA (1 µg) was subjected to Ribo-Zero depletion, fragmented, and then subjected to reverse transcription (RT). Double-stranded cDNAs were A-tailed and ligated with Illumina sequencing adapters. Subsequently, the ligated products were enriched by PCR and size-selected by agarose gel electrophoresis. The products (∼200 to ∼400 bp) were sequenced by the use of an Illumina Hi-seq 2500 platform with a paired 50-mer sequencing modality.
5
ChIP-seq Analysis of SHP Binding
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Mice were fasted overnight and injected with vehicle or FGF19, livers were collected 2 h later, and ChIP assays were performed using SHP antibody (sc-30169). Three sets of input and immunoprecipitated samples from three mice were pooled and 18 ng DNA was used for genomic sequencing using the Illumina/Solexa Genome Analyzer II (Biotechnology Center, University of Illinois at Urbana-Champaign). To validate the specificity of the SHP antibody for ChIP-seq, SHP binding to hepatic genes was analyzed by three independent ChIP assays for vehicle- or FGF19-treated WT and SHP-KO mice (Additional file 1 : Figures S1a, S5a).
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