RNA from bead-bound PCM-1(+) nuclei was collected by using the RNEasy Plus Micro kit (Qiagen, Hilden, Germany). Nuclear RNA sequencing (RNA-seq) libraries were constructed by using the Stranded RNA-seq Kit with Ribo Erase (Kapa Biosystems Inc.) with custom Y-shaped adapters. Paired-end 2×75 bp sequencing was performed for RNA-seq libraries with an Illumina Nextseq instrument (DNA Link). Reads were first mapped to the mouse genome (mm10) by using STAR (Dobin et al., 2013 ). Differential expression analysis was then carried out with DESeq2 (Love et al., 2014 (link)). Gene ontology analysis was performed by using Metascape [http://metascape.org ] (Tripathi et al., 2015 (link)), and displayed using GOplot (Walter et al., 2015 ). Gene set enrichment analysis using publicly available data(Uosaki et al., 2015 (link)) was performed by interrogating the top 200 most enriched transcripts in either adult hearts relative to embryonic (E12-14); or embryonic relative to adult against our RNA-seq dataset (enrichment score is relative to control) (Mootha et al., 2003 (link); Subramanian et al., 2005 (link); Uosaki et al., 2015 (link)).
Stranded rna seq kit
Manufactured by Roche
The Stranded RNA-Seq Kit is a laboratory instrument designed for the analysis of stranded RNA sequencing. It provides the necessary tools and reagents to prepare RNA samples for high-throughput sequencing, while maintaining the original strandedness of the RNA molecules.
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4 protocols using stranded rna seq kit
1
Cardiac transcriptome profiling by nuclear RNA-seq
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Stranded RNA-Seq Protocol with Multiplexing
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Nascent RNA sequencing by Bru-seq
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Nascent RNA sequencing by Bru-seq (Paulsen et al. 2014 (link)) was as described (Cortazar et al. 2019 (link)). HEK293 Xrn2 WT or D235A cells with doxycycline induction at 2 μg/mL for 24 h were incubated with 2 mM bromouridine in fresh medium plus doxycycline for 30 min. Labeled RNA was fragmented and then immunopurified with B44 monoclonal anti-BrdU (Gratzner 1982 (link)) immobilized on protein A Dynabeads with rabbit antimouse IgG. Immunoprecipitation of 30–50 μg of labeled RNA was performed in DEPC-treated 1× PBS, 0.2% (v/v) Triton X-100, and 1 mM DTT supplemented with RiboLock RNase inhibitor (EO0382). Beads were washed four times in the same buffer, and RNA was eluted in RNase-free water for 3 min at 95°C. RNA-seq libraries were made with the KAPA Biosciences stranded RNA-seq kit (KK8401). After filtering out rRNA, reads were mapped to the hg19 UCSC human genome (February 2009) with Bowtie2 version 2.3.2.
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RNA-Seq Library Preparation and Sequencing
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