Libraries were prepared following the PARS-seq protocol62 (link) (briefly described below), using the TruSeq Small RNA sample preparation kit of the Illumina. We ligated the 5′ adapter as described in the TruSeq Small RNA sample preparation protocol (Illumina) and stopped the reaction with 1 µl stop solution (a component of the TruSeq Small RNA sample preparation kit) in 28 C for 15 min. 3′ end treatment with Antarctic phosphatase was done as described in the directional mRNA-Seq sample preparation protocol, followed by its inactivation with ethanol precipitation. 3′ adapter ligation, RT and PCR amplification were performed as described in the TruSeq Small RNA sample preparation protocol. The cDNA was size selected and cleaned using E-Gel 4% agarose (Invitrogen) and Agencourt AMPure XP beads (Beckman Coulter). The library was quantified using Qubit (Invitrogen) and validated using Agilent 2100 Bioanalyzer. Libraries were sequenced on Illumina Hiseq2500 machine. Sequencing yields of the samples are detailed in Supplementary Table 1 .
Truseq small rna sample preparation kit
Manufactured by Illumina
Sourced in United States
The TruSeq Small RNA Sample Preparation Kit is a laboratory equipment product developed by Illumina. It is designed for the preparation of small RNA samples for sequencing. The kit provides a streamlined workflow for the isolation, purification, and library preparation of small RNA molecules from various sample types.
Automatically generated - may contain errors
Lab products found in correlation
Agilent 2100 bioanalyzer, by Agilent Technologies (43 mentions)
Hiseq 2500, by Illumina (34 mentions)
Hiseq 2000, by Illumina (27 mentions)
Trizol reagent, by Thermo Fisher Scientific (22 mentions)
Hiseq 2000 platform, by Illumina (21 mentions)
2100 bioanalyzer, by Agilent Technologies (17 mentions)
Hiseq 2500 platform, by Illumina (12 mentions)
Trizol, by Thermo Fisher Scientific (11 mentions)
Mirneasy mini kit, by Qiagen (11 mentions)
Hiseq 2000 system, by Illumina (9 mentions)
Mirvana mirna isolation kit, by Thermo Fisher Scientific (9 mentions)
Hiseq 4000, by Illumina (8 mentions)
Agilent bioanalyzer 2100 system, by Agilent Technologies (7 mentions)
Miseq platform, by Illumina (6 mentions)
Rna 6000 nano labchip kit, by Agilent Technologies (6 mentions)
Hiseq 2500 sequencer, by Illumina (5 mentions)
Superscript 2 reverse transcriptase, by Thermo Fisher Scientific (5 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (5 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (4 mentions)
Nextseq 500, by Illumina (4 mentions)
231 protocols using truseq small rna sample preparation kit
1
Illumina Small RNA Library Preparation
2
Determining 3'-Terminal U6 snRNA Sequence
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To determine the 3′-terminal sequence of U6 snRNA, we ligated the 3′ RNA adapter from the TruSeq Small RNA Sample Preparation Kit (Illumina, San Diego, CA, USA) onto 1 μg total RNA according to the supplier's protocol. Ligated RNA was reverse transcribed for 30 min at 42°C followed by 1 h at 50°C using a primer that introduces a primer binding site for subsequent amplification (Supplementary Table S4) and components of the TruSeq Small RNA Sample Preparation Kit (Illumina).The reverse transcriptase was inactivated by incubation of the sample at 70°C for 15 min. The cDNA was diluted 1:10 and U6 was amplified using a U6-specific primer and a primer complementary to the region introduced by reverse transcription (Supplementary Table S4) by Taq DNA polymerase (New England Biolabs, Ipswich, MA, USA). PCR amplicons were cloned into the pCR4TOPO vector according to the supplier's protocol (Life Technologies) and sequenced.
3
Illumina TruSeq Small RNA Library Prep
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We used the TruSeq Small RNA Sample Preparation Kit (Illumina) to prepare the libraries for further sequencing. We initially performed a phosphatase treatment incubating 16 µl of the digested samples for 30 min at 37 °C and 5 min at 65 °C mixed with 2.5 µl of 10X phosphatase buffer, 2.5 µl of nuclease-free water, 1 µl of RNAse inhibitor and 3 µl of Antarctic phosphatase (New England BioLabs Inc.). We then performed a kinase treatment adding 4 µl of T4 Polynucleotide Kinase (PNK, New England BioLabs Inc.), 5 µl of 10X PNK buffer, 10 µl of ATP 10 mM, 1 µl of RNAse inhibitor and nuclease-free water up to a total volume of 50 µl, and incubating the samples 1 h at 37 °C. Samples were then purified using RNeasy MiniElute Cleanup kit following manufacturer’s instructions (Qiagen) with a 10 μl RNase-free water final elution step. Samples were concentrated using a centrifugal evaporator Speed Vac® to a final volume of 5 μl and we started the TruSeq Small RNA Sample Preparation Kit (Illumina) protocol according to manufacturer’s instructions. After the final purification of the cDNA libraries, we performed quality control of each library using Agilent 2100 bioanalyzer with the DNA 1000 Kit (Agilent). Libraries were sequenced in single-reads with read lengths of 50 nucleotides in Illumina HiSeq2500 sequencers at the Genomics Unit of the CRG.
4
Small RNA Library Preparation for Sequencing
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We used the TruSeq Small RNA Sample Preparation Kit (Illumina) to prepare the libraries for further sequencing. We initially performed a phosphatase treatment incubating 16 µl of the digested samples for 30 min at 37 ºC and 5 min at 65 ºC mixed with 2.5 µl of 10X phosphatase buffer, 2.5 µl of nuclease-free water, 1 µl of RNAse inhibitor and 3 µl of Antarctic phosphatase (New England BioLabs Inc.). We then performed a kinase treatment adding 4 µl of T4 Polynucleotide Kinase (PNK, New England BioLabs Inc.), 5 µl of 10X PNK buffer, 10 µl of ATP 10 mM, 1 µl of RNAse inhibitor and nuclease-free water up to a total volume of 50 µl, and incubating the samples 1 hour at 37 ºC. Samples were then purified using RNeasy MiniElute Cleanup kit following manufacturer's instructions (Qiagen) with a 10 μl RNase-free water final elution step. Samples were concentrated using a centrifugal evaporator Speed Vac® to a final volume of 5 μl and we started the TruSeq Small RNA Sample Preparation Kit (Illumina) protocol according to manufacturer's instructions. After the final purification of the cDNA libraries, we performed quality control of each library using Agilent 2100 bioanalyzer with the DNA 1000 Kit (Agilent). Libraries were sequenced in single-reads with read lengths of 50 nucleotides in Illumina HiSeq2500 sequencers at the Genomics Unit of the CRG.
5
Small RNA Sequencing of Tumor Samples
6
Small RNA Sequencing from Total RNA
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Total RNA from four samples (two males and two females) samples was extracted using TRIzol (Life Technologies) as recommended by the manufacturer. RNA was dissolved in RNase-free water. For small RNA sequencing, I used the TruSeq Small RNA Sample Preparation Kit (Illumina) to generate the cDNA library using selected constructs of sizes 145–160 bp in a 6% PAGE gel, and precipitated in ethanol. DNA integrity was checked with TapeStation (Agilent). Samples were sequenced in-house with an Illumina MiSeq sequencing machine.
7
Small RNA Sequencing of Rice Samples
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One microgram of total RNA isolated from each rice sample was used to generate a library of small RNAs using a TruSeq Small RNA Sample Preparation Kit (Illumina) according to the sample-preparation instructions. Briefly, small RNA (<40 nt) was ligated with a single-stranded 3′-adapter and a bar-coded 5′-adapter. Ligated small RNA was reverse transcribed and amplified by PCR to generate individual DNA colony template library. Both libraries of Rby1-21 and Rby2-45 were used for 50 bp single-end sequencing by the Illumina HiSeq 2000 platform in two lanes.
8
Small RNA Library Preparation and Sequencing
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Total RNAs from the three different samples described above in the “Strains and growth conditions” section were used for small RNA library preparation and small RNA sequencing. Library preparation and sequencing was performed at GATC Biotech, Konstanz, Germany. Total RNA samples were separated by a 10% TBE-urea denaturing polyacrylamide gel electrophoresis (PAGE) and sRNAs ranging between 18 and 50 nt were used for library preparation using the Illumina TruSeq Small RNA Sample Preparation Kit. After reverse transcription and amplification, cDNA products were checked and measured with Bioanalyzer 2100. Sequencing was performed on an Illumina HiSeq 2000 platform. Trimming of TruSeq adapter sequences was performed with the program Cutadapt and only reads trimmed at their 3’ end were used for further studies [54 ]. Raw sequencing data from sRNA sequencing have been deposited in the NCBI sequence read archive (SRA) (http://www.ncbi.nlm.nih.gov/sra ) under the accession numbers SRR1705825 (RNA-Mix), SRR1706009 (∆ku70FRT2), and SRR1706010 (∆dcl2∆dcl1).
9
Genome-wide Pol II Occupancy and AGO4-RIP
10
Plasma Small RNA Sequencing Protocol
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Library preparation and sRNA sequencing was performed on each plasma sample using the Illumina platform, following the manufacturer’s protocol (Illumina Inc., San Diego, CA, USA). Small RNA libraries were constructed using a TruSeq Small RNA Sample Preparation kit (Illumina) according to the manufacturer’s instructions. Briefly, small RNA samples (18–30 nt) were gel-purified and ligated to the 5′ and 3′ adapters, followed by RT-PCR for cDNA library construction and incorporation of index tags. The cDNA library fragments were purified, separated on a PAGE gel and loaded on an Agilent Technologies 2100 Bioanalyzer to check size, purity, and concentration. Libraries were sequenced using Illumina HiSeq2000 at the BGI-Shenzhen (Shenzhen, Guangdong, China).
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