For cnRNA-seq, 2x107 mouse ESCs (untreated or treated with auxin or dTAG-13 compound for indicated times) were mixed with 8x106Drosophila SG4 cells in PBS. Nuclei were released in 1 mL HS Lysis buffer (50 mM KCl, 10 mM MgSO4.7H20, 5 mM HEPES, 0.05% NP40, 1 mM PMSF, 3 mM DTT, 1x PIC) for 1 min at room temperature, and recovered by centrifugation at 1000 g for 5 min at 4°C, followed by three washes with ice-cold RSB buffer (10 mM NaCl, 10 mM Tris pH 7.4, 3 mM MgCl2). Pelleted nuclei were resuspended in 1 mL of TRIzol reagent and RNA was extracted according to the manufacturer’s protocol, followed by treatment with the TURBO DNA-free Kit. Quality of RNA was assessed using 2100 Bioanalyzer RNA 6000 Pico kit (Agilent). Next, rRNA was depleted using the NEBNext rRNA Depletion kit (NEB). RNA-seq libraries were prepared using the NEBNext Ultra (for PRC1deg) or Ultra II (for PRC2deg) Directional RNA Library Prep kit (NEB), indexed using NEBNext Multiplex Oligos (NEB), polled and sequenced as 80 bp (PRC1deg) or 40 bp (PRC2deg) paired-end reads on the Illumina NextSeq 500 in biological triplicates.
Directional rna library prep kit
Manufactured by New England Biolabs
The Directional RNA Library Prep Kit is a tool designed for the preparation of directional RNA libraries for next-generation sequencing. It provides a streamlined workflow for the efficient conversion of total RNA into a high-quality sequencing library, ensuring the preservation of the original RNA strand orientation.
Automatically generated - may contain errors
Lab products found in correlation
Ribo zero kit, by Illumina (2 mentions)
Nebnext multiplex oligos, by New England Biolabs (1 mentions)
2100 bioanalyzer rna 6000 pico kit, by Agilent Technologies (1 mentions)
Ultra 2, by New England Biolabs (1 mentions)
Nebnext rrna depletion kit, by New England Biolabs (1 mentions)
Nextseq 500, by Illumina (1 mentions)
3 protocols using directional rna library prep kit
1
Simultaneous RNA-seq profiling of mouse ESCs and Drosophila SG4 cells
2
RNA Sequencing of Isolated Nuclei
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Nuclei were isolated by centrifugation for 0.5 min from GM12878 and EB3_2 clones following lysis in 0.5% NP40, 140 mM NaCl, 10 mM Tris-HCl (pH 7.4), and 1.5 mM MgCl2. Nuclear RNA was isolated using Trizol reagent using the manufacturer’s instructions, followed by DNase treatment to remove possible genomic DNA contamination. Briefly, ribosomal RNAs were removed using the Ribo-Zero kit (Illumina), RNA was fragmented into 250–300 bp fragments, and cDNA libraries were prepared using the Directional RNA Library Prep Kit (NEB). Paired-end sequencing was done on a NovaSeq 6000 at the OHSU MPSSR core facility. Sequences were aligned to the human genome (hg19) using the STAR aligner78 (link) with default settings. Duplicate reads and reads with map quality below 30 were removed with SAMtools79 (link).
3
RNA-seq analysis of HTD114 cell nuclei
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Nuclei were isolated from HTD114 cells following lysis in 0.5% NP40, 140 mM NaCl, 10 mM Tris–HCl (pH 7.4), and 1.5 mM MgCl2. Nuclear RNA was isolated using TRIzol reagent using the manufacturer's instructions, followed by DNase treatment to remove possible genomic DNA contamination. RNA-seq was carried out at Novogene. Briefly, ribosomal RNAs were removed using the Ribo-Zero kit (Illumina), RNA was fragmented into 250–300 bp fragments, and cDNA libraries were prepared using the Directional RNA Library Prep Kit (NEB). Paired-end sequencing was done on a NovaSeq 6000. Triplicate samples were merged and aligned to the human genome (hg19) using the STAR aligner (Dobin et al. 2013 (link)) with default settings. Duplicate reads and reads with map quality below 30 were removed with SAMtools (Li et al. 2009 (link)).
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