Total RNA (600 ng) was ligated to 5 μM of 5′-adenylated, 3′-blocked adaptor (Universal miRNA Cloning Linker, New England BioLabs) with 280 units of T4 RNA ligase, Truncated KQ (New England BioLabs), 25% PEG 8000 and 1 μl of RNaseOUT (Life Technologies) in a 20-μl reaction at 25 °C for 16–24 h. After cleanup with RNA Clean and Concentrator columns (Zymo Research), followed by DNase treatment, cDNA was synthesized with 5 pmol of universal RT primer (Supplementary Table 6 ) and SuperScript III reverse transcriptase. PCR amplification was carried out using 5 μM of the TERC_L2 and universal RT or TERC_L3 and universal RT primer sets (Supplementary Table 6 ) with SsoAdvanced Universal SYBR Green Supermix (Bio-Rad). PCR products were directly analyzed on 2.5% agarose gels to visualize mature TERC and extended TERC transcripts or subjected to QIAquick PCR purification columns (Qiagen) for library preparation for deep sequencing. For Sanger sequencing, 3′ RACE PCR products were directly cloned into the pCR4_TOPO vector (Life Technologies), and individual clones were sequenced using the TERC_L2 or TERC_L3 primer.
Universal mirna cloning linker
Manufactured by New England Biolabs
The Universal miRNA Cloning Linker is a molecular biology tool designed for the specific cloning and identification of microRNA (miRNA) molecules. It provides a standardized adapter sequence that can be ligated to the 3' end of miRNA samples, enabling their downstream amplification and sequencing.
Automatically generated - may contain errors
Lab products found in correlation
T4 rna ligase truncated kq, by New England Biolabs (3 mentions)
Rnaseout, by Thermo Fisher Scientific (3 mentions)
Superscript 3, by Thermo Fisher Scientific (3 mentions)
Sybr gold, by Thermo Fisher Scientific (2 mentions)
Kapa hyper prep kit, by Roche (2 mentions)
Ampure xp bead, by Beckman Coulter (2 mentions)
Verso cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Kod one pcr master mix, by Toyobo (2 mentions)
Pgem t easy vector, by Promega (2 mentions)
Rna clean concentrator 25 column, by Zymo Research (2 mentions)
T4 rna ligase, by New England Biolabs (1 mentions)
Truncated kq, by New England Biolabs (1 mentions)
Ssoadvanced universal sybr green supermix, by Bio-Rad (1 mentions)
Rna clean and concentrator column, by Zymo Research (1 mentions)
Qiaquick pcr purification column, by Qiagen (1 mentions)
Solid system, by Thermo Fisher Scientific (1 mentions)
Nextseq 500 high output kit v2, by Illumina (1 mentions)
Nextseq 500 platform, by Illumina (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Smarter pcr cdna synthesis kit, by Takara Bio (1 mentions)
15 protocols using universal mirna cloning linker
1
RNA-seq Analysis of TERC Transcripts
2
Quantification of Human Telomerase RNA
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600 ng of RNA was ligated to 5 uM of 5′ adenylated, 3′ blocked adaptor (Universal miRNA cloning linker, NEB S1315S) with 250 units of T4 RNA ligase truncated KQ (NEB M0373S), 25% PEG 8000, and 1 μL RnaseOUT (ThermoFisher 10777019) in a 20 μL reaction at 25 degrees for 16 hours. Ligated RNA was cleaned up with RNA clean and concentrator columns (Clontech 740955.50) and DNase treatment, cDNA was synthesized with universal primer and SuperScript III (ThermoFisher 18080093). Amplification was carried out with Phusion (New England Biosystems M0530) and primer sets universal/TERCR1 (listed in the Key Resources Table ). PCR products were directly run on an 8% PAGE gel and visualized with SYBR Gold (ThermoFisher S-11494), or subjected to AMPure XP beads (Beckman Coulter A63881) for PCR cleanup and library preparation. Libraries were prepped using Kapa Hyperprep Kit (Kapa KK8504), quantified with Qubit and bioanalyzer, and run on Illumina miSeq at the Stanford Functional Genomics Facility. Reads were paired using fastq-join tool at Galaxy (http://usegalaxy.org ). Reads were binned into the various forms of hTR using custom python scripts (https://cmroake.people.stanford.edu/links-python-scripts ) and the number of reads in each bin was normalized to total hTR reads.
3
Small RNA Library Preparation
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All raw and processed data are available at the NCBI Gene Expression Omnibus (GEO) with accession number GSE96999.
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3′ Preadenylated DNA linker (NEB Universal miRNA Cloning Linker, S1315S, dissolve into 50 µM) 5′-/rApp/CTGTAGGCACCATCAAT/NH2/-3′
5′ RNA linker (50 µM stock) 5′-/Biosg/rArCrArCrGrArCrGrCrUrCrUrUrCrCrGrArUrCrU-3
Reverse transcription primer (50 µM stock) 5′-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT (Important: The “RAN” represents a customizable randomer sequence that can be used to remove PCR duplicates later in the data analysis. Six-base randomers are used in the experiments, but longer is recommended for higher sequencing depth.)
Illumina compatible barcoded PCR primers i7: 5′-caagcagaagacggcatacgagat (Barcodes are customizable. The protocol is established using paired-end sequencing with dual barcodes. Important: When demultiplexing, the i7 barcode sequence needs to be specified as reverse complement to what is in
All raw and processed data are available at the NCBI Gene Expression Omnibus (GEO) with accession number GSE96999.
4
Preadenylated DNA Linker Ligation
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Preadenylated DNA linkers were made in-house as previously described (Hafner et al. 2008 (link)). We have used two different linkers successfully. The first linker was derived from Applied Biosystems SOLID system; the reverse SOLID sequence was preadenylated. Linker two was a modified version of NEB's universal miRNA cloning linker (Cat. #S1315S). We added 9 nt to the 3′ end of this linker to increase the Tm for the subsequent PCR reaction(s). We obtained similar results with both sets of linkers. Concatemerization of the preadenylated linker is prevented by blocking with 3′ end by addition of either a dideoxy nucleotide or an amine group. The amine blocker is equally effective and less expensive (Vigneault et al. 2008 (link)).
One and one-half micrograms of total RNA in a total volume of 10 µL was denatured for 10 min at 65°C and then cooled on ice for 2 min. Of note, 0.75 µg of preadenylated linker was added, followed by 200 units of truncated KQ T4 RNA ligase 2 (NEB Cat. # M0373S), using the buffer supplied by the manufacturer. Twenty units of RiboLock RNAse inhibitor was added to inhibit degradation. Ligation was carried out in the absence of ATP for 16 h at 16°C. After ligation the RNA was purified by phenol-chloroform extraction, followed by one extraction with chloroform and recovered by ethanol precipitation in the presence of glycoblue.
One and one-half micrograms of total RNA in a total volume of 10 µL was denatured for 10 min at 65°C and then cooled on ice for 2 min. Of note, 0.75 µg of preadenylated linker was added, followed by 200 units of truncated KQ T4 RNA ligase 2 (NEB Cat. # M0373S), using the buffer supplied by the manufacturer. Twenty units of RiboLock RNAse inhibitor was added to inhibit degradation. Ligation was carried out in the absence of ATP for 16 h at 16°C. After ligation the RNA was purified by phenol-chloroform extraction, followed by one extraction with chloroform and recovered by ethanol precipitation in the presence of glycoblue.
5
Mosquito Small RNA Sequencing
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RNA was extracted from pools of five mosquitoes using TRIzol. Small RNAs of 18-33 nt in length were purified from a 15% acrylamide/bisacrylamide (37.5:1), 7 M urea gel as described previously.53 (link) Libraries were prepared using the NEBNext® Small RNA Library Prep Set for Illumina® (New England BioLabs), with the Universal miRNA Cloning Linker (New England BioLabs) as the 3’ adaptor and in-house designed indexed primers. Libraries were diluted to 4 nM and sequenced using an Illumina NextSeq 500 High Output kit v2 (75 cycles) on an Illumina NextSeq 500 platform.
6
Cloning and Sequencing of Small RNAs
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Total RNA was isolated using RNAzol RT from 293T cells 24 h after transfection. Universal miRNA Cloning Linker (#S1315S, New England Biolabs) was linked to the 3′-terminus of RNA using T4 RNA Ligase 2, truncated K227Q (#M0351S, New England Biolabs). Then, cDNA was synthesized using Verso cDNA Synthesis Kit (#AB1453, Thermo Fisher Scientific), and PCR was performed using KOD One PCR Master Mix (#KMM-101, TOYOBO). The resulting PCR fragments were cloned into pSP73 and verified by Sanger sequencing. Primers are listed in Additional file 10 : Table S5.
7
Gm4745 mRNA Tailing and Sequencing
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Each sample included 100 2-cell embryos. Total RNA was isolated using RNeasy Mini
Kit (Qiagen, Cat. No. 74106) and ligated overnight at 25°C with a 2
μM Universal miRNA Cloning Linker (NEB, Cat. No. S1315S) using 200 U T4
RNA ligase 2 truncated KQ (NEB, Cat. No. M0373S) in the presence of 25%
PEG 8000 and RNaseOUT. The ligated RNAs were purified using RNA Clean &
Concentrator-25 columns (Zymo Research, Cat. No. R1017), and reverse transcribed
using SuperScript III (Invitrogen; Cat. No. 18080200) and universal
RT+ linker primer (Supplementary Table S1 ). Gm4745 mRNA tails were
amplified using Gm4745-specific forward and universal
RT+ linker reverse primers (Supplementary Table S1 ). PCR products were visualized on
2% agarose gels (Supplementary Figure S3D ), extracted, cloned into pGEM-T easy
vectors (Promega), and sequenced by Sanger DNA sequencing.
Kit (Qiagen, Cat. No. 74106) and ligated overnight at 25°C with a 2
μM Universal miRNA Cloning Linker (NEB, Cat. No. S1315S) using 200 U T4
RNA ligase 2 truncated KQ (NEB, Cat. No. M0373S) in the presence of 25%
PEG 8000 and RNaseOUT. The ligated RNAs were purified using RNA Clean &
Concentrator-25 columns (Zymo Research, Cat. No. R1017), and reverse transcribed
using SuperScript III (Invitrogen; Cat. No. 18080200) and universal
RT+ linker primer (
amplified using Gm4745-specific forward and universal
RT+ linker reverse primers (
2% agarose gels (
vectors (Promega), and sequenced by Sanger DNA sequencing.
8
Quantification of Human Telomerase RNA
9
RNA-seq Library Preparation Protocol
10
Ligation Assay for Small RNA Detection
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For the ligation assay, ribosomal RNA and tRNA were first removed from 2 μg total RNA, using the ZYMO RNA Clean & Concentrator-5 kit (Zymo R1015). The rRNA-depleted RNA (5 μL) was then mixed with 1 μL 50 pmol Universal miRNA cloning linker (NEB S1315S; 5′ rAppCTGTAGGCACCATCAAT–NH2 3′) at 65°C for 5 min, and then ligated using T4 RNA Ligase 2, truncated KQ (NEB M0373S) at 16°C for 10 h. After ligation with 3′ adaptor, cDNA was synthesized by SMARTer PCR cDNA Synthesis Kit (TaKaRa 634926). The 3′ cDNA end of individual gene was amplified by nest PCR using gene specific forward primers and ligation reverse primer (ligation-R). PCR products were separated by agarose gel and sent for Sanger sequencing. Primer sequences are listed in Supplemental Table S3 .
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