Muscle samples were dissected on dry ice and immediately homogenized in QIAzol Lysis Reagent (QIAGEN, Germantown, MD, USA) to extract RNA. Small RNA sequencing was performed as previously described.11 (link),35 In brief, 3 (liver) or 1 μg (muscle) of RNA was ligated to 3′ Universal miRNA Cloning Linker (New England Biosciences, Ipswich, MA, USA) using T4 RNA ligase 1 (New England Biosciences, Ipswich, MA, USA) without ATP, then run on an 8M urea-15% polyacrylamide gel. Seventeen- to twenty-eight-nucleotide fragments were excised and ligated to 5′ barcodes again using T4 RNA ligase, then multiplexed and sequenced on an Illumina miSeq machine obtaining 50-bp single-end reads at the University of Washington Center for Precision Medicine. Linkers and barcodes were trimmed from the sequences; then the sequences were aligned to mouse miRNAs on miRBase (release 15)36 (link) using Bowtie version 0.12.7, allowing for two mismatches.37 (link) Hierarchical clustering of miRNAs was performed using the DESeq R package.38 (link) Small RNA sequencing data have been deposited in the NCBI Gene Expression Omnibus (GEO) repository with accession number GEO: GSE129896 .
T4 rna ligase
Manufactured by Illumina
Sourced in United States
T4 RNA ligase is an enzyme that catalyzes the formation of a phosphodiester bond between the 5' phosphate of one RNA molecule and the 3' hydroxyl of another RNA molecule. It is commonly used in molecular biology applications involving RNA manipulation and ligation.
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Lab products found in correlation
Superscript 2 reverse transcriptase, by Thermo Fisher Scientific (3 mentions)
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Hiseq 2500, by Illumina (3 mentions)
Hiseq 2000, by Illumina (2 mentions)
5 adapter, by Illumina (2 mentions)
Tobacco acid pyrophosphatase, by Illumina (2 mentions)
Miseq machine, by Illumina (1 mentions)
Qiazol lysis reagent, by Qiagen (1 mentions)
Miseq reagent kit v2, by Illumina (1 mentions)
Truseq small rna library prep kit, by Illumina (1 mentions)
Miseq platform, by Illumina (1 mentions)
Solexa sequencer, by Illumina (1 mentions)
Specialized adaptors to the 5 and 3 ends, by Illumina (1 mentions)
Sybr safe dna gel stain, by Thermo Fisher Scientific (1 mentions)
Mirpremier microrna isolation kit, by Merck Group (1 mentions)
3 adapter, by Illumina (1 mentions)
Truseq rna sample prep kit, by Illumina (1 mentions)
Truseq small rna sequencing protocol, by Illumina (1 mentions)
Hiseq v4 reagents, by Illumina (1 mentions)
Agilent high sensitivity dna kit, by Agilent Technologies (1 mentions)
14 protocols using t4 rna ligase
1
Small RNA Sequencing of Muscle and Liver Tissues
2
Arabidopsis Small RNA Sequencing Protocol
3
Small RNA Sequencing Library Prep
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Small RNA libraries were constructed using the TruSeq small RNA library prep kit (Illumina), following the manufacturer’s protocol. Briefly, 1 μg of total RNA from each sample was ligated to 3′ adaptor using T4 RNA ligase 2 deletion mutant (Epicentre) followed by ligation to 5′ adaptor using T4 RNA ligase (Illumina). The ligated fragment was reverse transcribed followed by PCR amplification (11 cycles). The amplified products were size fractionated on 6% Novex TBE PAGE gels (Life Technologies). A band corresponding to 145–150 bp was purified and denatured. Sequencing was performed on Illumina MiSeq platform using MiSeq reagent kit v2.
4
sRNA Extraction and Sequencing for Radish Stress Response
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Total RNA was extracted from the control (NaCl-free, CK) and salt-stressed (200 mM NaCl for 48 h, Na200) radish roots with TRIzol reagent (Invitrogen, USA) following the manufacturer’s instructions. Two sRNA libraries were constructed according to previously reported procedures [25 (link),57 (link)]. Briefly, sRNA fractions of 18–30 nt isolated and purified by 15% denaturing polyacrylamide gel electrophoresis were ligated with specialized adaptors to the 5’ and 3’ ends (Illumina) using T4 RNA ligase. They were then reverse transcribed to cDNA using SuperScript II Reverse Transcriptase (Invitrogen), followed by PCR amplification. The final PCR products were purified and subjected to deep sequencing using Solexa sequencer (Illumina) HiSeq2000 at the Beijing Genomics Institute (BGI), Shenzhen, China.
5
Small RNA Sequencing from Plant Shoot Apexes
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sRNA samples were isolated from shoot apexes of HT and HS plants by use of the mirPremier microRNA Isolation Kit (Sigma-Aldrich). sRNA fractions of 18 to 30 nt were purified from 15 % denaturing polyacrylamide TBE-Urea gels with SYBR Safe DNA gel stain (Invitrogen). The size-selected sRNAs were sequentially ligated to the 3′ and 5′ adapters with T4 RNA ligase 2 and T4 RNA ligase, respectively (Illumina Inc.). Reverse transcription was preformed with adapters, and then PCR amplification was performed as described in the Illumina protocol. The PCR products were purified by separation and subsequent elution from 10 % TBE urea polyacrylamide gel. The libraries from the two samples were sequenced by use of Illumina Hiseq2000, which generated paired-end reads of 100 nt (Yourgene Bioscience Co., Taiwan). The sRNA sequence data were deposited in Gene Expression Omnibus (GEO) under accession no. GSE 50546 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE50546 ).
6
Yeast cell RNA extraction and 5'RACE
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Yeast cells were grown to stationary phase and regrown to mid-exponential phase in SCD-URA medium (as was done prior to expression measurements by FACS; see above). Cells were aliquotted to 25 mL and centrifuged to pellet cells at 3000g for 8 min. The growth medium was removed, and total RNA was extracted using lyticase digestion followed by the TriReagent (MRC) RNA extraction protocol.
RNA libraries for transcription start site mapping (5′ end RNA-seq) were prepared as in Wurtzel et al. (2010) (link). In brief, RNA was incubated with tobacco acid pyrophosphatase (TAP, Epicentre) to treat 5′ ends, and 3′ ends were blocked using NaOI4. Illumina's 5′ adapter was ligated to the RNA with T4 RNA ligase (NEB). cDNA priming was done using a YFP gene-specific primer (GSP). Following cDNA synthesis, YFP amplicons were amplified for 18 cycles using a nested YFP GSP attached to an Illumina 3′ adapter and a 5′ Illumina adapter as forward primer.
RNA libraries for transcription start site mapping (5′ end RNA-seq) were prepared as in Wurtzel et al. (2010) (link). In brief, RNA was incubated with tobacco acid pyrophosphatase (TAP, Epicentre) to treat 5′ ends, and 3′ ends were blocked using NaOI4. Illumina's 5′ adapter was ligated to the RNA with T4 RNA ligase (NEB). cDNA priming was done using a YFP gene-specific primer (GSP). Following cDNA synthesis, YFP amplicons were amplified for 18 cycles using a nested YFP GSP attached to an Illumina 3′ adapter and a 5′ Illumina adapter as forward primer.
7
RNA-Seq and Small RNA Sequencing of Luffa Pulps
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Luffa pulps of same quantity of the three independent biological replicates from near-isogenic lines JAAS-BR and JAAS-BS were mixed for building transcriptome libraries with Illumina TruSeq RNA Sample PrepKit (Illumina, San Diego, CA, USA) according to the use instructions38 (link). RNAs were isolated with Trizol reagents (Invitrogen, Waltham, MA, USA) in line with manufacturer agreement.
For the construction of two small RNA libraries, we respectively used the extracted RNA from the pulp samples of the two lines. Briefly, we seperated the 18–30 nt long small RNAs before purifying them with 15% denaturing polyacrylamide gel electrophoresis and then used T4 RNA ligase to ligate them to Solexa adapters at their 5′ and 3′ ends (Illumina). The assembled small RNAs were reversally transcribed to cDNA, followed by PCR amplification. Both small RNAs and the paired-end transcriptome were deep sequenced with a HiSeq. 2000 Solexa sequencer (Illumina) in BGI (Beijing Genomics Institute).
For the construction of two small RNA libraries, we respectively used the extracted RNA from the pulp samples of the two lines. Briefly, we seperated the 18–30 nt long small RNAs before purifying them with 15% denaturing polyacrylamide gel electrophoresis and then used T4 RNA ligase to ligate them to Solexa adapters at their 5′ and 3′ ends (Illumina). The assembled small RNAs were reversally transcribed to cDNA, followed by PCR amplification. Both small RNAs and the paired-end transcriptome were deep sequenced with a HiSeq. 2000 Solexa sequencer (Illumina) in BGI (Beijing Genomics Institute).
8
Small RNA Sequencing of Plasma, Serum, and Cell Lines
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Sequencing libraries were generated using a maximum volume of RNA sample (7 μL) for each plasma and serum sample and an equal amount of RNA input (1,500 ng) was used for each cell line according to Illumina's TruSeq small RNA Sequencing Protocol. A 3′ adapter was ligated to the RNA template using a truncated T4 RNA ligase 2. Next, 5′ adapters were added, using T4 RNA ligase and ATP (Illumina). Single stranded cDNA was created using Superscript II Reverse Transcriptase (Invitrogen). The cDNA was then PCR amplified and PCR products were size‐selected for 145–160 bp. Quality of sequence libraries was validated using Agilent high sensitivity DNA kit (Agilent Technologies) by measuring size, purity, and concentration on an Agilent 2,100 Bioanalyzer. Sequencing was performed on Illumina HiSeq 2000 paired‐end 125‐cycle (PE125) run, using HiSeq v4 reagents (Illumina). After in silico adapter removal, 15‐125 nt fragments were mapped to the reference genome using several remote databases hosted by NCBI. Deep sequencing data was analyzed using sRNAToolbox.35
9
Aphid Small RNA Sequencing
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Total RNAs were isolated from pooled winged and wingless S. avenae samples using Trizol reagent (Ambion, USA) according to manufacturer’s instructions. The resulting total RNA quantity was estimated on a NanoDrop spectrophotometer (ND-2000, USA), and by densitometry following denaturing agarose gel analysis. RNA integrity was further assessed using the Bioanalyzer 2100 (Agilent, CA, USA). Total RNA of each sample was size-fractionated on 15% TBE polyacrylaminde gel. Small RNA (sRNA) populations of 15–50 nt were extracted, purified, and ligated to 3′ chimeric oligonucleotide adapters (5′-TGG AAT TCT CGG GTG CCA AGG -3′) and 5′ (5′-GTT CAG AGT TCT ACA GTC CGA CGA TC -3′) using T4 RNA ligase (Illumina, USA). Ligation reaction products were used as template for synthesize of single-stranded cDNA with SuperScript II Reverse Transcriptase (Illumina, USA), and subsequently PCR amplified using Illumina’s primer set for 15 cycles. Amplified cDNA products were gel purified and sequenced on an Illumina HiSeq 2500 (Illumina, San Diego, CA, USA) at LC Sciences (Houston, TX, USA) using the LC Bio service (Hangzhou, China).
10
Small RNA Sequencing of N. lugens
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For small RNA library construction, total RNA extraction from the N. lugens of SW morphs female adults for three developmental phases were pooled and prepared according to the TruSeq Small RNA Sample Preparation Kits (Illumina, San Diego, CA, USA). In brief, Solexa sequencing was performed as follows. For each library, small RNA fragments measuring 18–26 nt were collected using PAGE gel, then ligated to 3′ chimeric oligonucleotide adaptors (5′-TGGAATTCTCGGGTGCCAAGG-3′) and 5′ (5′-GTTCAGAGTTCTACAGTCCGACGATC-3′) to its end of the RNA pool using T4 RNA ligase (Illumina, San Diego, CA, USA). The adaptor-ligated small RNAs then were reverse transcribed and used as templates for cDNA synthesis using Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA, USA). The cDNAs were amplified using Illumina’s small RNA primer sets with 15 PCR cycles needed to produce the sequencing libraries. The purified PCR amplification products were sequenced on an Illumina HiSeq 2500 at LC-Bio Tech (Hangzhou, China) according to the manufacturer’s protocol. Single-end read of 50-bp length was obtained.
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