Briefly, 30 µL of vRNAs or 30 µL of IVT RNA mixes were purified and concentrated to 10 µL using Agencourt RNAClean XP SPRI beads (Beckman Coulter) and subjected to first strand cDNA synthesis using the Superscript III First-Strand Synthesis System for RT-PCR Kit (Thermo Fisher Scientific) and a mixture of random hexamers and U12–U13 specific primers (see above). The second cDNA strand was synthetized using 5 U of E. coli RNase H, 40 U of E. coli DNA polymerase, and 10 U of E. coli DNA ligase for 2 h at 16°C (New England Biolabs). Double-stranded cDNAs were purified using Agencourt RNAClean XP SPRI beads (Beckman Coulter) and quantified using the Quant-iT RNA Assay Kit (Thermo Fisher Scientific).
Agencourt rnaclean xp spri beads
Manufactured by Beckman Coulter
Sourced in United States
Agencourt RNAClean XP SPRI beads are magnetic beads used for the purification of RNA samples. They utilize Solid Phase Reversible Immobilization (SPRI) technology to selectively bind RNA molecules, allowing for efficient separation and purification.
Automatically generated - may contain errors
Lab products found in correlation
Quant it rna assay kit, by Thermo Fisher Scientific (1 mentions)
Superscript 3 first strand synthesis system for rt pcr kit, by Thermo Fisher Scientific (1 mentions)
E coli dna ligase, by New England Biolabs (1 mentions)
E coli rnase h, by New England Biolabs (1 mentions)
E coli dna polymerase, by New England Biolabs (1 mentions)
Nextseq 500, by Illumina (1 mentions)
High sensitivity dna kit, by Agilent Technologies (1 mentions)
Scriptseq index pcr primers, by Illumina (1 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (1 mentions)
Scriptseq v2 rna seq library preparation kit, by Illumina (1 mentions)
Ercc spike in control, by Thermo Fisher Scientific (1 mentions)
Nextera xt sample preparation kit, by Illumina (1 mentions)
Rnase inhibitor, by Promega (1 mentions)
Hiseq 1500, by Illumina (1 mentions)
Nextseq 500 sequencer, by Illumina (1 mentions)
Nextera xt library prep kit, by Illumina (1 mentions)
Lysis buffer, by Takara Bio (1 mentions)
Recombinant rnase inhibitor, by Takara Bio (1 mentions)
Qubit dsdna high sensitivity assay kit, by Thermo Fisher Scientific (1 mentions)
Hiseq 2000 platform, by Illumina (1 mentions)
5 protocols using agencourt rnaclean xp spri beads
1
RNA Purification and cDNA Synthesis
2
RNA-Seq Library Preparation and Sequencing
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After RNA samples have been quantified and analyzed they were DNase treated using a TURBO DNase 2 U µL−1 (Thermo Fisher Scientific) and cleaned using Agencourt RNAClean XP SPRI beads (Beckman Coulter). RNA-Seq libraries were then produced from these samples using a slightly modified ScriptSeq v2 RNA-Seq Library Preparation Kit (Illumina) ensuring use of unique index primers through ScriptSeq Index PCR Primers (Sets 1–4) 48 rxns/set (Illumina). Libraries were quantified by both a Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific) and by High Sensitivity DNA Kit (Agilent). The resulting molarity was then used to determine at what concentration samples should be pooled to (either 1, 2, or 4 nM). After a desired pooling concentration was chosen, the samples were diluted to that particular molarity and 2 µL of each sample dilution was added into a single tube and submitted to the Boston University Microarray and Sequencing Resource Core Facility. Whole transcriptome RNA sequencing was performed by a NextSeq 500 (Illumina) at high output (400 M reads) with 75 bp paired end sequencing read length. The data was then analyzed in-house through a proprietary Galagan Lab pipeline.
3
Single-Cell RNA-Seq Sample Preparation
4
Single-cell RNA-seq of Circulating Tumor Cells
5
RNA-seq Analysis of Nanoparticle Exposure
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Total RNA was extracted from cells harvested at 0 h, 6 h, 12 h, and 24 h of exposure to NMs (25 µg/mL) using the TRIZOL reagent (Life Technologies, Stockholm, Sweden) according to the manufacturer’s recommendations. Total RNA was quantified by NanoDrop™ (NanoDrop Technologies, ThermoScientific, Stockholm, Sweden) and RNA quality was assessed using the Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA). Three biological replicates of each sample were submitted for RNA sequencing [19 (link)]. In brief, the sequencing was performed using 1 μg total RNA following the Illumina® mRNA-Seq library preparation protocol (Illumina, San Diego, CA, USA). To this end, poly(A) RNA was isolated by two rounds of oligo (dT)25 Dynabeads™ (Invitrogen, Stockholm, Sweden) purification. Then, the chemically fragmented mRNAs were purified by Agencourt RNAClean XP SPRI beads (Agencourt-Beckman Coulter, Beverly, MA, USA) and converted to first strand cDNA, followed by second strand cDNA synthesis. The paired-end sequencing library was prepared from purified double stranded cDNA using the NEBNext® DNA Library Prep Kit (Illumina, San Diego, CA, USA). The purified ligated product was PCR amplified and the prepared libraries were quantified and quality-assessed and sequenced on the Illumina® HiSeq 2000 platform (Illumina, San Diego, CA, USA).
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