After RNA isolation, RNA-sequencing libraries were constructed using the Smart-Seq2 protocol from (Picelli et al., 2014 (link)), with modifications. Briefly, 8 ng of high-quality RNA was reverse transcribed using SuperScript II (Life Technologies, Cat# 18064–014) with a poly-dT anchored oligonucleotide primer, and a template switching oligonucleotide (TSO) primer that generated homotypic PCR primer binding sites. The cDNA underwent 10 rounds of PCR amplification using KAPA HiFi Hotstart (Kapa Biosystems, Cat# KK2601), followed by Ampure bead (Beckman Coulter, Cat# A63881) clean-up. The quality of the amplified cDNA was tested using Qubit nucleic acid quantitation (Life Technologies). 2ng of high-quality amplified cDNA was fragmented with the Tn5 transposase from the Illumina Nextera kit (Illumina, Cat# FC-131-1096) to a median size of ~500 bp. The fragmented libraries were amplified with indexed Nextera primers (Illumina, Cat# FC-131-1002) using 12 rounds of PCR. Final libraries were purified with Ampure beads and quantified on an Agilent Bioanalyzer. Libraries were pooled for sequencing on an Illumina NovaSeq with an SP flow cell (paired reads 2 × 150 bp).
Qubit nucleic acid quantitation
Manufactured by Thermo Fisher Scientific
The Qubit nucleic acid quantitation is a fluorometric-based quantitation tool that measures the concentration of DNA, RNA, or protein in a sample. It provides precise and sensitive quantification of nucleic acids or proteins.
Automatically generated - may contain errors
2 protocols using qubit nucleic acid quantitation
1
Smart-Seq2 RNA-seq Library Preparation
2
Smart-Seq2 RNA-seq Library Preparation
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After RNA isolation, RNA-sequencing libraries were constructed using the Smart-Seq2 protocol from (Picelli et al., 2014 (link)), with modifications. Briefly, 8 ng of high-quality RNA was reverse transcribed using SuperScript II (Life Technologies, Cat# 18064–014) with a poly-dT anchored oligonucleotide primer, and a template switching oligonucleotide (TSO) primer that generated homotypic PCR primer binding sites. The cDNA underwent 10 rounds of PCR amplification using KAPA HiFi Hotstart (Kapa Biosystems, Cat# KK2601), followed by Ampure bead (Beckman Coulter, Cat# A63881) clean-up. The quality of the amplified cDNA was tested using Qubit nucleic acid quantitation (Life Technologies). 2ng of high-quality amplified cDNA was fragmented with the Tn5 transposase from the Illumina Nextera kit (Illumina, Cat# FC-131-1096) to a median size of ~500 bp. The fragmented libraries were amplified with indexed Nextera primers (Illumina, Cat# FC-131-1002) using 12 rounds of PCR. Final libraries were purified with Ampure beads and quantified on an Agilent Bioanalyzer. Libraries were pooled for sequencing on an Illumina NovaSeq with an SP flow cell (paired reads 2 × 150 bp).
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