EmPC-seq: Accurate RNA-sequencing and Bioinformatics Platform to Map RNA Polymerases and Remove Background Error

Materials and Reagents

1. 1.5 ml tubes

2. Pipette tips

3. Cuvettes

4. 0.22 μm filter

5. W303 yeast cells (GenBank Number: JRIU00000000)

6. 1 M Dithiothreitol (DTT, ThermoFisher, catalog number: P2325 )

7. Diethyl pyrocarbonate (Sigma, catalog number: 40718 )

8. UltraPure^TM DNase/RNase-Free Distilled Water (ThermoFisher, catalog number: 10977015 )

9. Yeast Extract (Sigma, catalog number: Y1625 )

10. Peptone (Sigma, catalog number: P0556 )

11. D-(+)-Glucose (Sigma, catalog number: G8270 )

12. Adenine (Sigma, catalog number: A8626 )

13. N-Lauroylsarcosine sodium salt, Sarkosyl (Sigma, catalog number: L9150 )

14. Trizma^® hydrochloride (Sigma, catalog number: T5941 )

15. Potassium chloride (Sigma, catalog number: P9333 )

16. Magnesium chloride (Sigma, catalog number: M8266 )

17. Biotinylated Nucleotides (Jena Bioscience, catalog number: NU series )

18. RNase Inhibitor, Murine (NEB, catalog number: M0314L )

19. Diethyl pyrocarbonate, DEPC (Sigma, catalog number: 40718 )

20. Liquified Phenol (Sigma, catalog number: P9346 )

21. Sodium acetate (Sigma, catalog number: S2889 )

22. Ethylenediaminetetraacetic acid, EDTA (Sigma, catalog number: EDS )

23. Sodium dodecyl sulfate (Sigma, catalog number: L3771 )

24. Chloroform (Sigma, catalog number: C2432 )

25. GlycoBlue^TM Coprecipitant (ThermoFisher, catalog number: AM9515 )

26. Ethyl alcohol, Pure (Sigma, catalog number: E7023 )

27. Sodium hydroxide (Sigma, catalog number: S8045 )

28. Triton^TM X-100 (Sigma, catalog number: X100 )

29. Monarch^® RNA Cleanup Kit (NEB, catalog number: T2030L )

30. Dynabeads^TM M-280 Streptavidin (ThermoFisher, catalog number: 60210 )

31. Sodium chloride (Sigma, catalog number: S7653 )

32. TRIzol^TM Reagent (ThermoFisher, catalog number: 15596018 )

33. Ambion^TM T4 RNA Ligase (ThermoFisher, catalog number: AM2141 )

34. T4 Polynucleotide Kinase (NEB, catalog number: M0201S )

35. Polyethylene glycol 8000 (Sigma, catalog number: 1546605 )

36. Adenosine 5'-Triphosphate, ATP (NEB, catalog number: P0756S )

37. dNTP Mix (ThermoFisher, catalog number: 18427088 )

38. Random Hexamer Primer (ThermoFisher, catalog number: SO142 )

39. SuperScript^TM III First-Strand Synthesis System (ThermoFisher, catalog number: 18080051 )

40. NEBNext^® Ultra^TM II Directional RNA Second Strand Synthesis Module (NEB, catalog number: E7550L )

41. MinElute PCR Purification Kit Print (QIAGEN, catalog number: 28004 )

42. NEBNext^® Ultra^TM II DNA Library Prep Kit for Illumina^® (NEB, catalog number: E7645L )

43. Qubit^TM dsDNA HS Assay Kit (ThermoFisher, catalog number: Q32851 )

44. YEPD medium (see Recipes)

45. 2.5× Transcription buffer (see Recipes)

46. AES Buffer (see Recipes)

47. Beads washing buffer (see Recipes)

48. Binding washing buffer (see Recipes)

49. Low Salt washing buffer (see Recipes)

50. High Salt washing buffer (see Recipes)

51. DEPC-H₂O (see Recipes)

52. 1 M sodium acetate solution (see Recipes)

Equipment

1. Eppendorf^® Research^® Plus Pipettes (Eppendorf, catalog number: EP series )

2. MaxQ^TM 6000 Incubated/Refrigerated Stackable Shakers (ThermoFisher, catalog number: SHKE6000 )

3. Eppendorf BioPhotometer^® (Eppendorf, model: D30 )

4. Megafuge^® (Heraeus, model: 1.0R )

5. 5 Liter General Purpose Water Bath (PolyScience, catalog Number: WBE05A11B )

6. NEBNext^® Magnetic Separation Rack (NEB, catalog number: S1515S )

7. Roto-Shake Genie^® (Zymo Research, catalog number: S5008 )

8. ProFlex PCR System (ThermoFisher, catalog number: 4484075 )

9. 5200 Fragment Analyzer System (Agilent, catalog number: M5310AA )

Software

1. ProSize (Agilent, https://explore.agilent.com/Software-Download-Fragment-Analyzer-Prosize)

2. Python (version 2.7.12, https://www.python.org/)

3. Cython (version 0.23.4, https://cython.org/)

4. NumPy (version 1.11.0, https://numpy.org/)

5. SciPy (version 0.17.0, https://www.scipy.org/)

6. Burrows-Wheeler Aligner (version 0.7.17-r1188, http://bio-bwa.sourceforge.net/)

7. samtools (version 1.9, http://www.htslib.org/)

8. pysam (version 0.15.0, https://pysam.readthedocs.io/en/latest/installation.html)

9. matplotlib (version 2.2.2, https://matplotlib.org/)

Procedure

1. Prepare yeast cells for nuclear run-on assay

   1. Inoculate a single yeast colony in 5 ml YEPD medium.

   2. Incubate overnight in a 30 °C incubator with the rotation speed set as 200 rpm.

   3. Measure the optical density (OD_600nm) of each cell culture.

   4. Dilute and re-inoculate the yeast to 10 ml of YEPD medium with an OD₆₀₀ of 0.2.

   5. Incubate the yeast cells in a 30 °C incubator until they reach an OD₆₀₀ of 0.4-0.6.

      
      

2. Yeast nuclear run-on assay

   1. Centrifuge the yeast cells at 2,000 × g for 5 min at 4 °C and draw off the supernatant.

   2. Wash the cell pellet with 5 ml of ice-cold DEPC-H₂O by pipetting up and down.

   3. Re-centrifuge the yeast cells at 2,000 × g for 5 min at 4 °C and draw off the supernatant.

   4. Resuspend the cell pellet with 4.75 ml of ice-cold DEPC-H₂O.

   5. Add 250 μl of 10% Sarkosyl solution and gently mix the solution.

   6. Hold on ice for 20 min.

   7. Centrifuge the yeast cells at 400 × g for 5 min at 4 °C and draw off the supernatant.

   8. Resuspend the cell pellet with 120 μl of 2.5× Transcription Buffer, 6 μl of 0.1 M DTT, 3.5 μl of each kind of 1 mM biotin-NTP, and 3 μl of RNase Inhibitor to a total volume as 143 μl.

   9. Add 142 μl DEPC-H₂O and 15 μl of 10% Sarkosyl and gently mix the solution.

   10. Incubate the mixture at 30 °C for 5 min with gentle pipetting up and down at the half-time point (2.5 min).

       
       

3. Nascent RNA extraction

   1. Centrifuge the reaction mixture at 400 × g for 5 min at 4 °C and draw off the supernatant.

   2. Quickly resuspend the pellet in 500 μl of Liquified phenol.

   3. Add 500 μl of AES Buffer and pipette up and down to rupture cells.

   4. Incubate the mixture at 65 °C for 5 min with vortex once every minute.

   5. Incubate the mixture on ice for 5 min.

   6. Add 200 μl of chloroform to it and vortex for 30 s.

   7. Incubate the mixture at room temperature for 2 min.

   8. Centrifuge it at 14,000 × g for 5 min at 4 °C.

   9. Draw off the aqueous layer and put it into another new tubes (be careful to avoid the interface).

   10. Add 1 M sodium acetate solution to the aqueous layer to a final concentration of 200 mM.

   11. Split the aqueous RNA solution mixed with sodium acetate into two 1.5 ml tubes.

   12. Add 1 μl of GlycoBlue^TM Coprecipitant and 3× volume of 100% ethanol into the two 1.5 ml tube containing aqueous layer in Step C11 (this mixture can be stored overnight).

   13. Centrifuge the mixture at 14,000 × g for 30 min at 4 °C and draw off the supernatant (be careful to avoid disturbing the blue pellet).

   14. Wash the pellet with freshly prepared RNase-free 75% ethanol.

   15. Centrifuge it at 14,000 × g for 5 min at 4 °C and draw off the supernatant.

   16. Let the pellet dry for 5 min and resuspend it in 20 μl of DEPC-H₂O.

       
       

4. RNA fragmentation by Base Hydrolysis

   1. Heat denature the RNA solution at 65 °C for 40 s and place it on ice.

   2. Add 5μl of ice cold 1 N NaOH and incubate it on ice for 10 min.

   3. Add 25 μl of 1 M Tris-HCl (pH 6.8).

   4. Purify the RNA with Monarch^® RNA Cleanup Kit (10 μg) with elution volume of 20 μl.

   5. Add 1 μl RNase inhibitor.

      
      

5. Biotin-labeled nascent RNA pull-out

   1. Wash 30 μl of Streptavidin M280 beads with 500 μl of beads washing buffer by adding and mixing the washing buffer with the beads, setting on a magnet for 1 min and drawing off the supernatant.

   2. Wash the beads twice with 500 μl of 100 mM NaCl solution, the operation is the same as Step E1.

   3. Resuspend the beads in 50 μl of binding washing buffer.

   4. Mix the purified RNA in section D to 50 μl with binding washing buffer.

   5. Mix the RNA solution with the resuspended beads.

   6. Incubate the mixture at room temperature on a rotator for 20 min.

   7. Place the mixture on a magnet for 1 min and draw off the supernatant.

   8. Resuspend the beads in 500 μl of high salt washing buffer.

   9. Place the beads on a magnet for 1 min and draw off the supernatant.

   10. Repeat Steps E8-E10.

   11. Wash the beads twice with 500 μl of binding washing buffer.

   12. Wash the beads twice with 500 μl of low salt washing buffer.

   13. Resuspend the beads in 300 μl of TRIzol^TM solution.

   14. Incubate the mixture on ice for 3 min.

   15. Add 60 μl of chloroform to the mixture and vortex it thoroughly for at least 20 s.

   16. Centrifuge the mixture at 20,000 × g for 5 min at 4 °C.

   17. Purify the RNA in the aqueous layer (be careful to avoid the interface) with Monarch^® RNA Cleanup Kit (10 μg) with an elution volume of 20 μl.

       
       

6. Cyclization of RNA sample

   1. Heat denature the purified RNA at 65 °C for 1 min and place it on ice.

   2. Add the following reagents to 19 μl of the RNA solution: 4 μl of 10× T4 ligase I reaction buffer, 2 μl of T4 Ligase I enzyme, 2 μl of PNK enzyme, 1 μl of RNase inhibitor, 8 μl of 50% PEG8000 and 4 μl of 10 mM ATP solution.

   3. Incubate the mixture at 25 °C for 2 h or at 16 °C overnight.

   4. Purify the RNA in the mixture with Monarch^® RNA Cleanup Kit (10 μg) with elution volume of 20 μl.

      
      

7. Rolling-cycle reverse transcription

   1. Add the following reagents to 9 μl of the RNA solution: 4 μl of 10mM dNTPs solution, 4 μl of 50 ng/μl Random Hexamers and 3 μl of RNase-free water.

   2. Heat denature the reaction mix at 65 °C for 1 min and place it on ice for more than 2 min.

   3. Add the following materials into the reaction mix: 8 μl of 5× First-strand synthesis buffer, 4 μl of 0.1 mM DTT solution, 4 μl of SuperScript III enzyme and 8μl of water

      Note: The buffers and enzymes used here are from SuperScript^TM III First-Strand Synthesis System Kit.

   4. Incubate the mixture at 25 °C for 10 min and then incubate at 42 °C for 20 min.

   5. Purify the RNA in the mixture with Monarch^® RNA Cleanup Kit (10 μg) with an elution volume of 20 μl.

   6. Add 20 μl of RNase-free water to the RNA solution.

      
      

8. Second strand synthesis

   1. Add 38 μl of RNA solution with the following materials: 8 μl of 10× Second strand synthesis buffer, 4 μl of Second strand synthesis enzyme mix and 30 μl of RNase-free water

      Note: The buffers and enzymes used here are from NEBNext^® Ultra^TM II Directional RNA Second Strand Synthesis Module Kit.

   2. Incubate the reaction mix at 16 °C for 2 h with the thermocycler lid set at 50 °C.

   3. Purify the DNA in the mixture with the MinElute PCR Purification Kit.

      
      

9. Library preparation and submitting for NGS

   1. Measure the concentration of cDNA in step H3 by using Qubit^TM dsDNA HS Assay Kit.

   2. Prepare the sequencing library with the NEBNext^® Ultra^TM II DNA Library Prep Kit for Illumina^® according to the manufacturer’s instructions by end preparation, adaptor ligation, size selection and PCR enrichment.

   3. Submit the cDNA library to MiSeq Illumina platform with read length as single-end 300 base pairs for performing Next Generation Sequencing.

Data analysis

1. Use Ubuntu 16.04 LTS (Xenial Xerus) for running the script.

2. Download the script from GitHub (https://github.com/ustsam/Em-PC_seq).

3. Unzip the file.

4. Make sure all software in software section is installed.

5. Open a terminal and enter the script directory

6. Compile the code by typing “python setup_newreloc.py build_ext –inplace” in a terminal.

7. Call the function using the command in the terminal:

   
   

   “./run_noQsfilter.sh {PATH to the output directory} {PATH to the reference file} {PATH to the script directory} DUMMY 2 ${twice of the max readlength} ${PATH of the data file in gzipped form}.”

   
   

8. data.sam.gz can be found in the output directory. “data.sam.gz” contains all the transcripts after the consensus generation step. The file is in compressed sam format. To decompress the file, one can type the following command in a linux terminal:

   gunzip data.sam.gz

   
   

9. Run the command in the output directory to perform data analysis:

   
   

   “bash data_analysis.sh {PATH to the output directory} {PATH to the reference file} {PATH to the script directory} 1 {maximum depth per site} {minimum base quality} {minimum mapping quality} {number of simulation fastq files generated}.”

   
   

   Ambiguity is defined as the number of ways to map a transcript to the reference genome (Cheung et al., 2020). We used the strictest threshold, which is ambiguity=1, minimum mapping quality=30, and minimum base quality=30.

10. Run the following command to reproduce the analysis (assuming the script and output directory, as well as the reference fasta file are in the current directory):

    
    

    “bash data_analysis.sh ./ ./rDNA1.fasta ./ 1 500000 30 30 100.”

    
    

11. Run the following command to preprocess the output from “data_analysis.sh” script for plotting:

    
    “bash plot.sh {PATH to the output directory} {PATH to the reference file} {PATH to the script directory} {ambiguity threshold} {maximum depth per site} {minimum base quality} {minimum mapping quality} 1.”

    
    

12. The output of plot.sh files figures (.png format) are the following:

    1. “Distribution_NumberOfWaysToMap.png”: the distribution of the number of ways the transcripts can be mapped to the reference genome (ambiguity). The y-axis is the number of transcripts and the x-axis is ambiguity. An example is shown below (Figure 2).

       
       

       
       Figure 2. An example of a figure of the percentage of transcripts with a particular ambiguity. The ambiguity is the number of ways a transcript can be mapped to the reference genome. Ambiguity arises from the lack of information about the start of transcription due to the circularization step during the experiment.

       
       

    2. “MutationTypeSpectrum.png”: The mutational frequency for each type of mutation in the RNA transcript. The mutational frequency is the number of errors divided by the coverage of the corresponding reference base. For example: number of A °C errors divided by coverage of base A. An example is shown below (Figure 3).

       
       

       
       Figure 3. An example of a figure of the mutational spectrum. The mutational frequency of each type of substitution on the RNA chain is shown.

       
       

    3. “Muta_Frequency_inChrom_***.png”: The mutational frequency along the sites in the chromosome for the experimental and simulation data. “***” is the name of the chromosome. The transcription error-enriched genomic loci (TEEL) is shown as red dots. An example is shown below (Figure 4).

       
       

       
       Figure 4. An example of a figure of the mutational frequency across the genomic sites. The mutational frequency for each site in the chromosome is shown as the red lines. The background error obtained by simulation is shown as the gray line. The sites that are identified as TEEL are shown as red dots.

       
       

    4. “ErrorRate_per_PositionInTranscripts.png”: The error rate at each position in the transcript. The Position 0 corresponds to the 3’ end of the transcript. An example is shown below (Figure 5).

       
       

       
       Figure 5. An example of a figure of the error rate at each positional site in the transcript. The error rate for each position from the experimental data is shown as red dots. The error rate for each position from the simulation data is shown as blue dots. The x-axis is the position in the transcript, where position 0 corresponds to the 3’ end of the transcript. The increased error rate could be correlated by pausing of the RNA Polymerase, yet the order of event could not be determined in this experiment. Two scenarios could fit the observed data, either RNA Polymerase pauses to fix the error or RNA Polymerase pauses due to the misincorporation.

       
       

13. If one would prefer to use other software tools, the output text files to plot the figures are:

    1. “Distribution_of_Ambiguity.txt” can be used to plot the figure described in step 12a.

    2. “MutationTypeSpectrum.txt” can be used to plot the figure described in step 12b.

    3. “MutationalFrequency_Exp_chrom_***.txt” can be used to plot the figure described in step 12c and “MutationalFrequency_Sim_chrom_***.txt” are the files containing the Mutational Frequencies per site in a chromosome for experimental and simulation, respectively. “MutationalFrequency_TEEL_chrom_***.txt” contains the mutational frequency of the sites considered as TEEL. The first column is the position in the chromosome and the second column is the mutational frequency.

    4. “MutationalFrequency_PerPositionInTranscript.txt” can be used to plot the figure described in step 12d. The first column is the position in the transcript. The second and third column is the average and standard deviation of the simulation data. The fourth and fifth column is the average and standard deviation of the experimental data. The average and standard deviation of experimental data are calculated from error rate binomial distribution estimated by maximum likelihood.

Notes

1. RNA digestion: There are several alternative methods of RNA fragmentation since the type of RNA and the expected final size impact the sequencing technology to be used. For instance, ultra-sonication is widely used to fragment DNA for NGS sequencing libraries and can be optimized for RNA fragmentation as well, however, the size of the product is around 100-200 nt and careful consideration is needed when trying to obtain more than 2 tandem repeats during rolling-circle reverse transcription. Meanwhile, another biological digestion method using RNase III enzyme can fragment RNA molecules into smaller sizes (60-120 nt) but the recovery rate is relatively low (around 10%).

2. RNA electrophoresis: We suggest collecting RNA samples at each step for troubleshooting. RNA electrophoresis can assay the size and amount of RNA. For small amounts of RNA larger than 200 bp but smaller than 6,000 bp, a Fragment Analyzer (Agilent) can be used since it needs no more than 2 ng of RNA and can generate a comprehensive size/amount spectrum of the RNA samples.

3. RNA purification: Monarch^® RNA Cleanup Kit is used to replace ethanol precipitation as it is quicker and easier to operate with multiple samples. Ethanol precipitation can still be used if RNA cleanup kit is not available but improper phase separation of aqueous layer may result in contamination with TRIzol^TM.

4. Library Preparation: In this protocol we use the NEBNext^® UltraTM II DNA Library Prep Kit for Illumina^® to prepare the cDNA library for sequencing. There are also other alternative kits for library preparation such as Ovation^® Ultralow V2 DNA-Seq Library Preparation Kit and KAPA HTP/LTP Library Preparation Kits. While the steps of preparing cDNA libraries using different library preparation kits are similar, there are several points that are needed to be mentioned. Firstly, the dilution of the adaptor and the PCR enrichment cycles are different among these kits according to the specific mass of cDNA used for library preparation. For example, NEBNext^® Ultra^TM II DNA Library Prep Kit for Illumina^® needs 25-fold dilution of adaptor and a 10-cycles PCR if the sample mass is around 1 ng. Secondly, different library preparation kits have designated index primers, it should be considered that each kit might have their own requirement about the index combination based on the number of cDNA samples. For instance, NEBNext^® Ultra^TM II DNA Library Prep Kit for Illumina^® suggests several unique combinations when the number of cDNA samples is less than 7.

5. DEPC Operation: Solid DEPC is toxic and harmful. Thus, there should be proper eye-shield, face-shield, full-face respirator, gloves and chemical hood when performing any operation with solid DEPC.

6. Data analysis: Due to circularization, the information about the transcriptional direction and starting point of the transcripts are unknown. The scripts prepared are specifically used to treat rRNA transcripts, where they assume that all the transcripts are transcribed in the negative direction, i.e., the starting position is the 3’ end of the transcript. The consensus generation step (steps 7 and 8) is general for all raw data and does not depend on the transcription direction. However, starting from step 9, we assume negative direction of transcription, thus changes are required to generalize the script for other transcripts. The following files will need to be modified: “data_analysis.sh”, “pysam_make_pileup.py”, “simulation.py”, “binomial_distribution.py”, “plotting.py” and “plotting_preprocess.py”. In addition, ambiguity occurs due to re-localization during data treatment. Ambiguity is defined as the number of ways a transcript can be mapped to the reference genome (Cheung et al., 2020). The script assumes the strictest requirement, where only the transcripts with ambiguity equals to one are considered in analysis.

Recipes

1. 1 L YEPD medium

   1. Add 10 g of Yeast Extract

   2. Add 20 g of peptone

   3. Add 850 ml dH₂O

   4. Dissolve 20 g glucose in 100 ml dH₂O in a new bottle

   5. Dissolve 40 mg adenine in 50 ml dH₂O in a new bottle

   6. Autoclave above reagents at 120 °C for 15 min

   7. Add glucose and adenine solutions into the pre-medium after it cools down

2. 2.5× Transcription buffer

   1. Add Tris-HCl (pH=7.7) to the final concentration as 50 mM

   2. Add KCl to the final concentration as 500 mM

   3. Add MgCl₂ to the final concentration as 12.5 mM

   4. Add DEPC-H₂O to a total volume as 50 ml

3. AES Buffer

   1. Add Sodium Acetate (pH=5.3) to the final concentration as 50 mM

   2. Add EDTA to the final concentration as 10 mM

   3. Add SDS to the final concentration as 1% (w/v)

   4. Add DEPC-H₂O to a total volume as 50 ml

4. Beads washing buffer

   1. Add NaOH to the final concentration as 0.1 N

   2. Add NaCl to the final concentration as 50 mM

   3. Add DEPC-H₂O to a total volume as 50 ml

5. Binding washing buffer

   1. Add Tris-HCl (pH = 7.4) to the final concentration as 10 mM

   2. Add NaCl to the final concentration as 300 mM

   3. Add Triton^TM X-100 to the final concentration as 0.1% (v/v)

   4. Add DEPC-H₂O to a total volume as 50 ml

6. Low Salt washing buffer

   1. Add Tris-HCl (pH = 7.4) to the final concentration as 5 mM

   2. Add Triton^TM X-100 to the final concentration as 0.1% (v/v)

   3. Add DEPC-H₂O to a total volume as 50 ml

7. High Salt washing buffer

   1. Add Tris-HCl (pH = 7.4) to the final concentration as 50 mM

   2. Add NaCl to the final concentration as 2 M

   3. Add Triton^TM X-100 to the final concentration as 0.5% (v/v)

   4. Add DEPC-H₂O to a total volume as 50 ml

8. DEPC-H₂O

   1. Add 0.1% (v/v) DEPC to RNase-free Water

   2. Mix overnight and then autoclave

   3. Filter-sterilize the solution with a 0.22 μm filter

   4. Store in room temperature for up to one year

9. 1 M sodium acetate solution

   1. Add 4.1 g solid sodium acetate

   2. Add DEPC-H₂O to a total volume as 50 ml

Acknowledgments

This work was supported by Shenzhen Science and Technology Innovation Committee (JCYJ20170413173837121), Innovation and Technology Commission (ITCPD/17-9), the Hong Kong Research Grant Council (HKUST C6009-15G, AoE/P-705/16, T13-605/18-W) to X.H; GRF 16301319 to P.P.C; National Institute of Health GM25232 to J.L. NSFC (31922088); Hong Kong ITC (ITCPD/17-9, ITS/480/18FP); Hong Kong RGC (26102719, N_HKUST606/17, C6002-17GF, C7065-18GF, R4017-18) to J.W. B.J. was supported by the Institute of Advanced Studies (IAS) postdoctoral fellowship.

This protocol was derived from Cheung et al. (2020)

Competing interests

All authors declare no conflicts of interests.

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