Universal mirna cloning linker
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.
Lab products found in correlation
15 protocols using universal mirna cloning linker
RNA-seq Analysis of TERC Transcripts
Quantification of Human Telomerase RNA
Small RNA Library Preparation
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.
Preadenylated DNA Linker Ligation
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.
Mosquito Small RNA Sequencing
Cloning and Sequencing of Small RNAs
Gm4745 mRNA Tailing and 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.
Quantification of Human Telomerase RNA
RNA-seq Library Preparation Protocol
Ligation Assay for Small RNA Detection
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