Total RNA was extracted from 22Rv‐1‐30e stable lines with and without doxycycline induction and used for RNA Sequencing performed by LC Sciences. First, RNA integrity was checked using Agilent Technologies 2100 Bioanalyzer (Agilant Technologies, Santa Clara, CA, USA). Ribosomal RNA was depleted, and the sequencing library was prepared using Illumina's TruSeq‐stranded‐total‐RNA‐sample preparation protocol (Illumina, San Diego, CA, USA). This includes RNA fragmentation, reverse transcription using random primers, dUTP incorporation, A‐ tailing, adapter ligation, strand degradation and PCR enrichment. Illumina's NovaSeq 6000 sequencing (Illumina) was used to perform paired‐end sequencing. RNA quality control and library preparation were performed by LC Sciences (Houston, TX, USA).
Truseq stranded total rna sample preparation protocol
Manufactured by Illumina
Sourced in United States
The TruSeq Stranded Total RNA Sample Preparation Protocol is a library preparation kit designed for RNA sequencing. It enables the generation of stranded RNA-seq libraries from total RNA samples. The protocol preserves the strand orientation of the original RNA molecule, allowing for the identification of the original sense and antisense transcripts.
Automatically generated - may contain errors
Lab products found in correlation
Hiseq 2500, by Illumina (2 mentions)
Rnalater, by Thermo Fisher Scientific (2 mentions)
Novaseq 6000 sequencing, by Illumina (1 mentions)
2100 bioanalyzer high sensitivity dna chip, by Agilent Technologies (1 mentions)
Novaseq 6000 sequencing system, by Illumina (1 mentions)
Animal tissue rna purification kit, by Norgen Biotek (1 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
Bioanalyzer rna nano 600 kit, by Agilent Technologies (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Qiazol, by Thermo Fisher Scientific (1 mentions)
Mirneasy kit, by Qiagen (1 mentions)
Agilent rna 6000 nano kit, by Agilent Technologies (1 mentions)
6 protocols using truseq stranded total rna sample preparation protocol
1
Transcriptome Analysis of 22Rv-1-30e Cell Lines
2
Transcriptomic Analysis of Ribodepleted RNA
3
Strand-specific RNA Sequencing Protocol
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In total, 1 μg of total RNA was ribodepleted as described in the Rho-seq section and analyzed by microchip electrophoresis as described above. Overall, 125 ng of ribodepleted samples were prepared for strand-specific NGS with the TruSeq Stranded Total RNA Sample Preparation Protocol (Illumina). The library was sequenced on Illumina HiSeq 2500 (paired-end reads, 2× 50 nt). Reads were mapped on the genome using TopHat2. The quantification was made with featureCounts function on R and the differential expression analysis was performed with DESeq2. The genes following these criteria were considered as differentially expressed; false discovery rate <10% and the absolute value of the fold change >1.5.
4
Sponge Transcriptome Response to Algae
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Sponges were grown from gemmules in 1X Strekal’s to the stage where a functioning osculum had developed. To triplicate samples of these sponges (∼20–30 sponges per treatment), we added live algal cells (130,000 Chlorella ml−1) or no algae as treatments. Tissue was collected after 24 h of exposure to algae, washed several times to remove algae from the surrounding water and surfaces, and either stored at −80 °C after RNAlater treatment (Thermo Fisher Scientific, Waltham, MA, USA) or processed immediately for RNA. Total RNA was isolated using the animal tissue RNA purification kit (Norgen Biotek, Thorold, Ontario, Canada). Total RNA was sent to LC Sciences (Houston, TX, USA) where RNA integrity was checked with Agilent Technologies 2100 Bioanalyzer (Agilent, CA). Ribosomal RNA was removed at LC Sciences using Ribo-Zero ribosomal RNA reduction, followed by fragmentation with divalent cation buffers in elevated temperature. Sequencing libraries were prepared by LC Sciences following Illumina’s TruSeq-stranded-total-RNA-sample preparation protocol (Illumina, San Diego, CA, USA). Quality control analysis and quantification of the sequencing library were performed using Agilent Technologies 2100 Bioanalyzer High Sensitivity DNA Chip. Paired-ended sequencing was performed on Illumina’s NovaSeq 6000 sequencing system by LC Sciences.
5
RNA Isolation and Sequencing Workflow
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RNA was isolated with TRIzol reagent (Invitrogen, Thermo Fisher Scientific Inc.) according to manufacturer's instructions. RNA quality was assessed by Agilent Bioanalyzer RNA 600 Nano Kit and RIN values >9 were accepted. Random fragmentation, cDNA synthesis and library generation were performed according to TruSeq Stranded Total RNA Sample Preparation protocol (Illumina). Libraries were subjected to sequencing, mapped onto the human reference genome and the resulting unique reads were counted across annotated protein-coding transcripts. For details, see Supporting File 1: Supporting Methods .
6
Liver Total RNA Extraction and Sequencing
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Total RNA was isolated from mouse livers with Qiazol (Invitrogen, #79306) and purified with the miRNeasy kit (Qiagen, #217004). The amount of RNA was quantified with the Agilent RNA 6000 Nano Kit (Agilent Technologies, #5067-1511). RNA-sequencing libraries were prepared from mouse liver total RNA using the Illumina TruSeq Stranded Total RNA sample preparation protocol following the manufacturer's instructions.
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