Whole-cell genomic DNA was isolated from either the JRB310 wild-type strain, the wild-type sexual progeny (exconjugants) or dcl-1 mutants, dcl-123 and dcl-141, and rdrp mutants, rdrp13, rdrp17, and rdrp25 using the Nucleospin Tissue DNA Extraction kit (Macherey-Nagel). One microgram of total genomic DNA was used to prepare Illumina libraries using a standard manufacturer's protocol and run on Illumina HiSeq 2500. Small RNAs were gel-purified from total RNA and loaded on denaturing 7 M Urea, 15% polyacrylamide gel. Of note, 17- to 25-nt small RNAs or 30- to 50-nt RNA (from dcl-141) were gel purified and cloned using the Illumina Truseq small RNA library construction kit. For RNA-seq, total RNA was isolated from wild-type reference strain JRB310 and two mutant strains, dcl-141 and rdrp13. Poly(A) RNA enrichment was performed using the polyA Spin mRNA Isolation Kit (NEB). Enriched RNA was reverse transcribed using Superscript III (ThermoFisher) and strand-specific libraries were constructed using published methods (Zhang et al. 2012 (link)). Barcoded libraries were mixed and sequenced.
Truseq small rna library construction kit
Manufactured by Illumina
Sourced in United States
The TruSeq Small RNA Library Preparation Kit is a laboratory equipment product designed for the construction of small RNA libraries. It enables the preparation of small RNA samples for sequencing on Illumina platforms. The kit includes reagents and protocols for the purification, adapter ligation, reverse transcription, and PCR amplification of small RNA molecules.
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Lab products found in correlation
2 protocols using truseq small rna library construction kit
1
Genomic DNA Extraction and RNA-Seq Analysis
2
Small RNA Library Generation and Sequencing
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Small non-coding RNA libraries were generated from the aforementioned tissues using the TruSeq small RNA library construction kit according to the manufacturer's protocol (Illumina, San Diego, CA, U.S.A.). Briefly, the 3′ and 5′ adapters were ligated to small RNAs from the total RNA sample followed by reverse-transcription PCR amplification. PCR was performed with two primers that annealed to the ends of adapters and contained indexes. Subsequently, the libraries with unique indexes were pooled together; the cDNA was gel-purified using a TBE PAGE gel and then concentrated by ethanol. Following a successful library quality control by qPCR, flow cell cluster generation was performed using a cBot. Single-end multiplexed sequencing was done using the Illumina GAIIx platform with the total of 36 cycles.
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