Chapter 2 - Springer Link

Chapter 2 Measuring Nascent Transcripts by Nascent-seq Fei Xavier Chen, Stacy A. Marshall, Yu Deng, and Sun Tianjiao Abstract A complete understanding of transcription and co-transcriptional RNA processing events by polymerase requires precise and robust approaches to visualize polymerase progress and quantify nascent transcripts on a genome-wide scale. Here, we present a transcriptome-wide method to measure the level of nascent transcribing RNA in a fast and unbiased manner. Key words Nascent-seq, Transcription, Pol II, mRNA, Next-generation sequencing

1  Introduction Transcription is a remarkably dynamic process during which the information in DNA is copied into RNA. High-throughput sequencing has revealed different categories of noncoding RNAs transcribed in coordination with or independent of protein-coding messenger RNA (mRNA) [1, 2]. Transcription of these coding and noncoding RNA molecules requires a precise and efficient collaboration of numerous transcription factors to regulate polymerase recruitment, transcriptional initiation, pause release, elongation, and termination [3, 4]. For transcription of mRNA and a subset of noncoding RNAs, transcription-coupled RNA processing events (e.g., 5′ capping, co-transcriptional splicing, and polyadenylation) add an additional layer of complexity to the regulation of transcription [5]. Thus, approaches to precisely measuring the level of nascent transcripts on genome-wide scale are essential for a full understanding of the dynamic process of transcriptional control. With the help of high-throughput sequencing, various strategies have been developed to measure the global transcriptional activity. In principle, they can be grouped into two categories. The first, Global Run-On sequencing (GRO-seq) [6] and Precision nuclear Run-On sequencing (PRO-seq) [7], is built on the measurement of nascent RNA extended by run-on assay with modified nucleotides in vitro. However, the treatment with sarcosyl and resumption of transcription Steven R. Head et al. (eds.), Next Generation Sequencing: Methods and Protocols, Methods in Molecular Biology, vol. 1712, https://doi.org/10.1007/978-1-4939-7514-3_2, © Springer Science+Business Media, LLC 2018

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elongation with labeled nucleotides in vitro may introduce biases and misrepresent the native transcription profiles. The second, Native Elongating Transcript sequencing (NET-seq) [8–10] and nascent RNA sequencing (nascent-seq) [11–14], relies on the extraordinary stability of the DNA–RNA–RNA polymerase ternary complex [15]. In NET-seq, nascent transcripts are enriched and purified by immunoprecipitation of actively transcribing endogenous or tagged RNA polymerase on chromatin. For immunoprecipitation of endogenous polymerase, antibodies that recognize polymerases in different states (e.g., Pol II with different phosphorylation at CTD) with the same or similar efficiency are essential to depict the global transcription profiles in a minimally biased manor. Nascent-seq, which we describe in this chapter, was developed on the basis of the fact that engaged polymerase containing nascent RNA tightly associates with chromatin through a stringent wash with high salt and urea buffer [16]. Nascent RNA is then purified from this washed chromatin. Compared with other approaches measuring nascent transcripts, nascent-seq is much simpler in terms of nascent RNA isolation and library preparation, which enables a fast and efficient measurement of nascent transcripts. The process of nascent-seq roughly contains four procedures: isolation of cell nuclei, purification of nascent RNA, library preparation, and next-generation sequencing. Nuclei isolation and nascent RNA purification can be finished within 1 day. Library preparation is usually split into 2 days (the first day for cDNA preparation and the second day for library preparation). Sequencing takes no more than ~1–2 h to set up and runs for ~11–13 h for reads of 50 bp in length.

2  Materials 2.1  Equipment and Supplies

1. Allegra X-14R Centrifuge (Beckman Coulter, Carlsbad, CA, USA). 2. Microfuge 20R Centrifuge (Beckman Coulter). 3. Wheaton Dounce tissue grinder, 7 mL. 4. UV-Vis Spectrophotometer, e.g., Nanodrop 2000 (ThermoFisher Scientific, Waltham, MA, USA). 5. Nutator. 6. 1.7 mL Microtubes. 7. 8-Tube Strips with Attached Domed Caps, 0.2 mL PCR tubes. 8. Magnetic Rack. 9. Thermal Cycler. 10. 2100 Bioanalyzer or Tapestation (Agilent Technologies, Santa Clara, CA, USA). 11. Qubit® 2.0 Fluorometer, Assay Tubes, and dsDNA HS Assay Kit (ThermoFisher Scientific).

Measuring Nascent Transcripts by Nascent-seq

2.2  Reagents and Buffers

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1. Phosphate-buffered saline (PBS). 2. Hypotonic Buffer: 10 mM HEPES, pH = 7.9; 10 mM KCl; 2 mM MgCl2; 1 mM DTT, add before use; 1× protease inhibitor (Sigma, St. Louis, MO, USA), add before use. 3. Nuclei Wash Buffer: 10 mM HEPES, pH = 7.9; 250 mM Sucrose; 1 mM DTT, add before use; 1× protease inhibitor, add before use; 50 U/mL RNase inhibitor (ThermoFisher Scientific). 4. 2× NUN Buffer (prepared in RNase-free water): 40 mM HEPES, pH = 7.9; 15 mM MgCl2; 0.4 mM EDTA; 600 mM NaCl; 2% v/v Nonidet P40. 5. 1× NUN Buffer: 50% volume of 2 M fresh urea solution; 50% volume of ice-cold 2× NUN Buffer; 50 U/mL RNase inhibitor. 6. Trypan Blue Solution, 0.4% (ThermoFisher Scientific). 7. TRIzol Reagent (ThermoFisher Scientific). 8. Chloroform. 9. Isopropyl alcohol. 10. 75% ethanol (in RNase-free water). 11. DNase I (RNase-free) (New England Biolabs, Ipswich, MA, USA). 12. High Sensitivity DNA Kit (Agilent Technologies). 13. RNA 6000 Nano Kit (Agilent Technologies). 14. RNase-free water. 15. 10 mM Tris–HCl, pH 8.0. 16. 200 mM Tris–HCl, pH 7.0. 17. 1 M Sodium hydroxide solution. 18. AMPure XP-PCR Purification (Beckman Coulter). 19. RNAClean XP with Scalable throughput (Beckman Coulter). 20. SuperScript II Reverse Transcriptase (ThermoFisher Scientific). 21. TruSeq® Stranded Total RNA LT—(with Ribo-ZeroTM Human/Mouse/Rat) (Illumina, San Diego, CA, USA).

3  Methods 3.1  Preparation of Cell Cultures

1. Culture 1–5 × 107 cells for each nascent RNA-seq sample (see Note 1). 2. For adherent cells, harvest the cells with trypsin-EDTA or by scraping in PBS; for suspension cells, pellet the cells by spinning at 300 × g for 5 min. Discard the supernatant. 3. Resuspend the pellet with 10 mL of ice-cold PBS and transfer the cells to a 15 mL conical tube. Spin at 300 × g for 5 min at

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4 °C to pellet cells. Repeat this wash two more times. Cell pellet can be used directly for nuclei isolation or be flash-frozen in liquid nitrogen and stored at −80 °C until use, with the former recommended to avoid the interference in nuclei isolation by the freezing and thawing of cells. 3.2  Isolation of Cell Nuclei

1. Resuspend the cell pellet in 10 mL of ice-cold Hypotonic Buffer with protease inhibitor and DTT. Incubate the cell suspension on ice for 15 min. 2. Centrifuge the cell suspension at 200  ×  g for 10 min at 4 °C. Discard the supernatant. 3. Resuspend the pellet in 5 mL of cold Hypotonic Buffer with protease inhibitor and DTT. 4. Transfer cell suspension into a precooled 7 mL Dounce tissue grinder. Homogenize the suspension with tight pestle with ~10 strokes. Check cell disruption under the microscope with Trypan Blue staining. Aim for ~90% disruption (Disrupted cells can take up the dye, unbroken cells cannot). Do not over-disrupt the cells. 5. Pellet the nuclei by spinning at 600 × g for 10 min at 4 °C. Discard the supernatant that contains the cytoplasmic fraction. 6. Resuspend the pellet with 5 mL of ice-cold Nuclei Wash Buffer with DTT, protease and RNase inhibitor. Spin at 1500 × g for 5 min at 4 °C and discard the supernatant. Repeat this wash once more.

3.3  Purification of Nascent RNA

1. Prepare 1× NUN buffer by mixing 50% volume of 2 M urea solution (fresh and filtered) and 50% volume of ice-cold 2× NUN Buffer supplemented with DTT, protease and RNase inhibitor (see Notes 2 and 3). 2. Suspend the pellet and disrupt the nuclei with 1 mL of 1× NUN buffer by pipetting up and down vigorously for 10–15 times. 3. Immediately transfer the suspension into a 1.5 mL RNase-free Eppendorf tube. Incubate on a nutator or rotating wheel for 5 min at 4 °C. 4. Pellet the chromatin by spinning at 1000 × g for 3 min at 4 °C. Carefully remove the supernatant using pipette instead to vacuum to avoid losing the chromatin pellet (see Note 4). 5. Resuspend the chromatin pellet carefully with 1 mL of 1× NUN buffer. Incubate on a nutator or rotating wheel for 5 min at 4 °C. Remove the supernatant carefully with pipette. Repeat this wash twice more. For the last spinning, use 5000 × g for 5 min at 4 °C (see Note 5). 6. Add 1 mL of TRIzol Reagent into chromatin pellet and vortex for 30 s. The chromatin pellet should be still visible.

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7. Incubate the homogenized sample for 5 min at 50 °C to permit complete dissolving of chromatin pellet and dissociation of the nucleoprotein complex vortex for 30 s. 8. Process the rest steps of RNA extraction following standard protocol of RNA purification using TRIzol. Resuspend the RNA pellet in 85 μL of RNase-free water. 9. Add 10 μL of 10× DNase I Reaction Buffer and 5 μL of DNase I (RNase-free) into RNA solution and mix well by pipette. Leave at room temperature for 20 min. 10. Add 300  μL of RNase-free water into RNA solution. 11. Add 200 μL of chloroform into RNA solution. Process the rest steps of RNA extraction following standard protocol of RNA purification using TRIzol. Resuspend the RNA pellet in 40 μL of RNase-free water. Leave the RNA solution on ice. 12. Measure the concentration of purified RNA with NanoDrop. A260/A280 is ~2.0. Low A260/A280 values (