TGIRT-seq Protocol for the Comprehensive Profiling of Coding and Non-coding RNA Biotypes in Cellular, Extracellular Vesicle, and Plasma RNAs

Materials and Reagents

Use reagents and solutions that are RNA grade and nuclease free. Store solutions in frozen aliquots to avoid repeated freezing and thawing.

1. 1.5 ml DNA LoBind microcentrifuge tubes (Eppendorf, catalog number: 022431021)

2. 2 ml DNA LoBind microcentrifuge tubes (Eppendorf, catalog number: 022431048)

3. Ep Dualfilter T.I.P.S LoRetention 0.1-10 μl (Eppendorf, catalog number: 0030078632)

4. Ep Dualfilter T.I.P.S LoRetention 2-100 μl (Eppendorf, catalog number: 0030078659)

5. Ep Dualfilter T.I.P.S LoRetention 20-300 μl (Eppendorf, catalog number: 0030078675)

6. Ep Dualfilter T.I.P.S LoRetention 50-1,000 μl (Eppendorf, catalog number: 0030078683)

7. 8 × 0.2 ml PCR reaction tube strip with attached flat cap (Simport, catalog number: T3202N)

8. Dithiothreitol (DTT), 1 M (Thermo Fisher Scientific, catalog number: P2325)

9. dNTPs mix, 25 mM each (Thermo Fisher Scientific, catalog number: R1122). Dilute to 20 mM each with RNase-free water before use

10. TGIRT-III enzyme (InGex, catalog number: TGIRT50)

    Note: Store TGIRT-III enzyme received from a supplier at -80°C until ready for use. Store opened tubes at -20°C. TGIRT-III may lose activity after 3 months of storage at -20°C (Behrens et al., 2021).

11. MinElute Reaction Cleanup kit (Qiagen, catalog number: 28204 or 28206) or MinElute PCR Purification kit (QIAGEN, catalog number: 28004 or 28006)

12. 5’ DNA adenylation kit (New England Biolabs, catalog number: E2610S/L)

13. Oligo Clean & Concentrator kit (Zymo Research, catalog number: D4060/4061)

14. Thermostable 5’ app DNA/RNA ligase (New England Biolabs, catalog number: M0319S/L)

15. Phusion high-fidelity PCR master mix with HF buffer (Thermo Fisher Scientific, catalog number: F531S/L)

16. AMPure XP (Beckman Coulter, catalog number: A63881)

17. High sensitivity DNA kit (Agilent, catalog number: 5067-4626)

18. (Optional) RNA 6000 Pico kit (Agilent, catalog number: 5067-1513)

19. (Optional) Small RNA kit (Agilent, catalog number: 5067-1548)

20. Oligonucleotides: All oligonucleotides should be HPLC-purified RNase-free grade

    1. R2 RNA

       5’-rArArG rArUrC rGrGrA rArGrA rGrCrA rCrArC rGrUrC rUrGrA rArCrU rCrCrA rGrUrC rArC/3SpC3/-3’

       Note: Other blockers such as 3’ Amino Modifier C6 dT(3AmMC6T) from IDT can also be used.

    2. R2R DNA

       5’-GTG ACT GGA GTT CAG ACG TGT GCT CTT CCG ATC TTN-3’ (N = equimolar A, T, G, C)

       Note: The R2R DNA used in the current TGIRT-seq protocol differs from that used in earlier versions. It has a single nucleotide change (insertion of the underlined T residue at the -3 position from the 3' end) that strongly decreases formation of R1R-R2R adapter-dimers during the ligation step (Xu et al., 2019). A complementary A residue is inserted at the corresponding position in R2 RNA (underlined A; see above).

    3. R1R DNA

       5’-/5Phos/GAT CGT CGG ACT GTA GAA CTC TGA ACG TGT AG/3SpC3/-3’

       Note: The Read 1 (R1) sequence corresponds to the small RNA sequencing primer site used in the NEBNEXT Small RNA Library Prep Set for Illumina sequencing.

    4. 6N unique molecular identifier (UMI) R1R DNA

       5’-/5Phos/NNN NNN GAT CGT CGG ACT GTA GAA CTC TGA ACG TGT AG/3SpC3/-3’

       Note: UMI nucleotides (machine-mixed equimolar A, C, G, and T residues, denoted N) are added at the 5’ end of the R1R sequence. The number of N nucleotides can be changed to suit the complexity of the samples being sequenced and the number of duplicates expected after PCR.

    5. Illumina barcode PCR primer (P5)

       5’-AAT GAT ACG GCG ACC ACC GAG AT BARCODE C TAC ACG TTC AGA GTT CTA CAG TCC GAC GAT C-3’

       Note: The barcode sequence in the primer is the sense strand (e.g., ATCACG in the primer for TruSeq Barcode 01 (TSBC01) on the Illumina website). This barcode is optional but recommended to provide unique dual indices (UDI) for libraries sequenced on a NovaSeq instrument.

    6. Illumina barcode PCR primer (P7)

       5’-CAA GCA GAA GAC GGC ATA CGA GAT BARCODE GTG ACT GGA GTT CAG ACG TGT GCT CTT CCG ATC T-3’

       Note: The barcode sequence in the primer should be the reverse complement of the barcode listed on the Illumina website (e.g., CGTGAT in the primer for TSBC01).

21. UltraPure DNase/RNase-free distilled water (Thermo Fisher Scientific, catalog number: 10977015) or equivalent from other companies or in house sources

22. Trizma hydrochloride solution (Tris-HCl), pH 7.5, 2 M (Sigma-Aldrich, catalog number: T2944)

23. EDTA, 0.5 M, pH 8.0, RNase-free (Thermo Fisher Scientific, catalog number: AM9260G)

24. Sodium chloride solution, 5 M, RNase-free (Thermo Fisher Scientific, catalog number: AM9760G)

25. Magnesium chloride solution, BioUltra for molecular biology, 2 M (Sigma-Aldrich, catalog number: 68475-100ML-F)

26. RNA Century^TM-Plus Markers (e.g., Thermo Fisher Scientific, catalog number: AM7145)

27. AMPure XP beads (Beckman Coulter, catalog number: A63880)

28. mirVana miRNA Isolation kit (Thermo Fisher Scientific, catalog number: AM1560)

29. Total Exosome RNA and Protein Isolation kit (Thermo Fisher Scientific, catalog number: 4478545)

30. TRIzol LS Reagent (Thermo Fisher Scientific, catalog number: 10296010)

31. Turbo DNase (Thermo Fisher Scientific, catalog number: AM2238)

32. DNase I (Zymo Research, catalog number: E1010)

33. Exonuclease I (Lucigen, catalog number: X40520K)

34. Illumina Ribo-Zero Plus rRNA Depletion kit (Illumina, catalog number: 20040526)

35. NEBNext Magnesium RNA Fragmentation Module (New England Biolabs, catalog number: E6150S)

36. RNA Clean & Concentrator-5 (Zymo Research, catalog number: R1013)

Equipment

1. -20°C freezer

2. T100 96-well PCR thermal cycler (Bio-Rad, catalog number: 1861096), Veriti 9044 60-well thermal cycler (Applied Biosystems, catalog number: 4384638), or equivalent thermal cycler with a heated lid and stable temperature control

3. Microcentrifuge (Eppendorf, catalog number: 2231000768)

4. DynaMag-2 Magnet (Thermo Fisher Scientific, catalog number: 12321D)

5. 2100 Bioanalyzer instrument (Agilent, catalog number: G2939BA)

6. Chip priming station (Agilent, catalog number: 5065-4401)

Software

1. 2100 Expert software/upgrade (Agilent, catalog number: G2946CA)

   Note: The Bioanalyzer software should be bundled with the instrument. If not, an upgraded version is purchasable from Agilent.

Procedure

1. Preparation of 10× Starter Duplex (Table 1)

   
   

   Table 1. Components needed for 10× (1 μM) Starter Duplex

   Components	Volume (final concentration)
   10 μM R2 RNA	5 μl (1 μM)
   10 μM R2R hand-mixed DNA	5 μl (1 μM)
   10× TE buffer (100 mM Tris-HCl, pH 7.5, 10 mM EDTA)	5 μl (10 mM Tris-HCl, pH 7.5, 1 mM EDTA)
   Nuclease-free H2O	to 50 μl

   
   

   1. Prepare 10 µM R2R DNA containing an equimolar ratio of A, C, G, and T residues at the 1-nt DNA overhang position by hand-mixing equal volumes of four separate R2R DNA oligonucleotides with 3' A, C, G, or T residues (10 µM each) in a 1.5-ml Eppendorf Lobind microcentrifuge tube or equivalent and vortex. Aliquot the stock solution and store at -20°C until use. A volume of 50 μl of 10× Starter Duplex is sufficient for 25 reactions (2 μl per reaction).

      Note: R2R DNA with unequal ratios of 1-nt 3’ DNA overhang nucleotides can be used to mitigate template-switching biases in TGIRT-seq (Mohr et al., 2013; Xu et al., 2019) ,

   2. Set up the above reaction components in a sterile PCR tube for annealing of oligonucleotides to form the Starter Duplex.

   3. Incubate the mixed 10× Starter Duplex components at 82°C for 2 min in a pre-heated thermocycler.

   4. Cool to 25°C with a 10% ramp or at a rate of 0.1°C/s.

      Note: The annealed R2 RNA/R2R DNA Starter Duplex should be prepared freshly each time before the template-switching reaction. Discard any left over after use.

      
      

2. Template-switching reverse transcription reaction

   1. Set up the following reaction components in a sterile PCR tube adding the TGIRT-III enzyme last (Table 2).

      
      

      Table 2. Components needed for template-switching reverse transcription reaction

      Components	Volume (final concentration)
      RNA sample	0.5-50 ng or <100 nM
      5× Reaction Buffer (1 M NaCl, 25 mM MgCl2, 100 mM Tris-HCl, pH 7.5)	4 μl (200 mM NaCl, 5 mM MgCl2, 20 mM Tris-HCl, pH 7.5)
      10× DTT (100 mM; avoid excessive freezing and thawing)	2 μl (10 mM)
      10× Starter Duplex (after annealing)	2 μl (100 nM final)
      TGIRT-III enzyme (10 μM)	1 or 2 μl (500 nM or 1 μM final)
      Nuclease-free H2O	to 19 μl

      
      

      Notes:

      1. Because TGIRT enzymes can also template switch to DNA, RNA samples should be DNase treated prior to TGIRT-seq. Minimum RNA Inputs for DNase-treated RNA samples are: 2 ng rRNA-depleted, chemically fragmented cellular RNA; 20-50 ng rRNA-depleted, unfragmented cellular RNAs; 500 pg plasma RNA; 2 ng RNA extracted from highly purified extracellular vesicles. Measure low RNA concentrations with a Qubit or Bioanalyzer. See Notes 1-4 at the end of the text for further details regarding preparation of RNA samples for TGIRT-seq.

      2. We use 200 mM NaCl in this protocol instead of 450 mM NaCl used in earlier versions to increase the efficiency of template switching without prohibitively increasing multiple sequential template switches, which result in artifactual fusion reads. See also Note 5 below.

      3. Do a pilot experiment to determine the optimal enzyme concentration for your samples using an Agilent Bioanalyzer or equivalent instrument to monitor library quality and quantity.

      4. A template-switching reverse transcription reaction using the TGIRT-III enzyme to a commercial RNA ladder (e.g., Thermo Fisher Scientific, catalog number: AM7145) can be carried through the procedure as a positive control.

   2. Pre-incubate at room temperature for 30 min, then initiate template switching and reverse transcription by adding 1 μl of 20 mM dNTPs (an equimolar mixture of 20 mM each dATP, dCTP, dGTP, and dTTP).

   3. Incubate at 60°C for 15 min for whole cell, EV, or plasma RNAs, 5-10 min for mature miRNAs, or up to 60 min for long or heavily modified RNAs. The optimal incubation time should be determined experimentally for different RNA templates.

   4. Add 1 μl of 5 M NaOH and incubate at 95°C for 3 min.

   5. Cool to room temperature and neutralize with 1 μl of 5 M HCl.

      Note: Steps B4 and B5 are needed because TGIRT-III binds very tightly to nucleic acids. Alkaline hydrolysis degrades the RNA template and enzyme but not the cDNA products.

   6. Add 78 μl of nuclease-free water to bring to a final volume of 100 μl.

   7. Clean up the cDNAs with a MinElute Reaction Cleanup kit (QIAGEN, catalog number: 28204) or a MinElute PCR Purification kit (QIAGEN, catalog number: 28004) and elute in 10 μl of QIAGEN elution buffer.

      Note: Incubate the column with the elution buffer at room temperature before centrifugation to maximize recovery.

      The procedure can be interrupted here, with the cDNAs stored at -20°C.

   8. Proceed with R1R adenylation, thermostable ligation, and Phusion PCR amplification.

      
      

3. R1R 5' DNA adenylation using the New England Biolabs kit (New England Biolabs, catalog number: E2610S/L)

   Note: If the adenylated oligonucleotide is purchased from a commercial supplier, proceed to the ligation step.

   1. Set up the following reaction components in a sterile PCR tube (Table 3):

      
      

      Table 3. Components needed for 5' DNA adenylation

      Components	Volume
      10× reaction buffer (DNA Adenylation Buffer)	2 μl
      1 mM ATP	2 μl
      100 μM 5’phos/3’SpC3 R1R DNA	1 μl
      Mth RNA Ligase	2 μl
      Nuclease-free water	to 20 μl

      Note: The New England Biolabs kit includes 10× DNA Adenylation buffer, 1 mM ATP, and Mth RNA ligase.

      
      

   2. Incubate at 65°C for 1 h.

   3. Incubate at 85°C for 5 min to inactivate the enzyme.

   4. Clean up with an Oligo Clean & Concentrator kit (Zymo Research, catalog number: D4060) and elute in 10 μl of nuclease-free water for a final concentration of 10 μM 5’-end adenylated R1R DNA.

      Note: If required for a large number of TGIRT-seq libraries, we recommend scaling up the adenylation reaction by doing multiple (e.g., 6× to 8×) 20-μl reactions with the same amounts of enzyme and oligonucleotide, and then combining products for clean-up, as higher elution volume helps with efficient recovery of adenylated oligonucleotides. After clean-up, recovered material should be 80-100 ng/μl for R1R and 100-120 ng/μl for 6N UMI R1R, measured with a Nanodrop spectrophotometer using the ssDNA setting. Adenylation can be monitored using an Agilent Bioanalyzer with a Small RNA kit. The adenylated oligonucleotide can be stored at -20°C for up to two weeks.

      
      

4. Thermostable ligation of a 5’ adenylated adapter (New England Biolabs, catalog number: M0319S/L)

   1. Set up the following reaction components in a sterile PCR tube (Table 4):

      
      

      Table 4. Components needed for thermostable ligation of 5’ adapter

      Components	Volume
      10× reaction buffer (NEBuffer 1)	2 μl
      50 mM MnCl2	2 μl
      cDNA from template-switching reaction	Up to 10 μl
      Thermostable 5’ AppDNA/RNA Ligase	2 μl
      10 μM 5’-end adenylated R1R DNA	4 μl
      Nuclease-free water	To 20 μl if using less than 10 μl cDNA

      
      

   2. Incubate at 65°C for 1-2 h.

      Note: We recommend 2 h ligation. However, 1 h ligation can be used if the starting material is abundant.

   3. Add 80 μl of nuclease-free water for a final volume of 100 μl.

   4. Clean up the ligated cDNAs with a MinElute Reaction Cleanup kit (QIAGEN, catalog number: 28204) and elute in 23 μl of QIAGEN elution buffer (incubate the column with elution buffer at room temperature before centrifugation to maximize recovery.

      Note: The procedure can be interrupted here with the ligated cDNAs stored at -20°C.

   5. Proceed with Phusion PCR amplification.

      
      

5. PCR amplification (Thermo Fisher Scientific, catalog number: F531S/L)

   1. Set up the following reaction components in a sterile PCR tube (Table 5):

      
      

      Table 5. Components needed for PCR amplification

      Components	Volume (final concentration)
      2× Phusion High-Fidelity PCR Master Mix in HF buffer	25 μl
      10 μM Illumina P5 primer	1 μl (200 nM)
      10 μM Illumina P7 Primer	1 μl (200 nM)
      cDNA from thermostable ligation	Up to 23 μl
      Nuclease-free water	To 50 μl, if using less than 23 μl cDNA

      
      

   2. PCR cycles:

      1. Denaturation 98°C for 5 s.

      2. Up to 12 cycles of 98°C for 5 s, 60°C for 10 s, 72°C for 15-30 s/kb, hold at 4°C.

         Notes:

         1. Minimizing the number of cycles of PCR amplification decreases bias and duplicate reads. Twelve PCR cycles is generally satisfactory for TGIRT-seq with RNA inputs indicated in Section B. The number of cycles can be increased or decreased for different RNA inputs and library complexities.

         2. The procedure can be interrupted here and the PCR products stored at -20°C.

   3. Use AMPure XP beads (Beckman Coulter, catalog number: A63880) to deplete adapter-dimers and enrich for desired DNA sizes in the library. The default ratio is 1.4× v/v (70 μl of beads/50 μl of PCR reaction). The ratio of beads to sample volume can be adjusted depending on the size profile of RNAs being sequenced.

   4. Check library quality by analyzing 1 μl on a Bioanalyzer with a High Sensitivity DNA Analysis kit (Figure 2).

      
      

      
      Figure 2. Bioanalyzer traces of representative TGIRT-seq libraries obtained by using an Agilent Bioanalyzer with a High Sensitivity DNA kit. A. Ribo-depleted and chemically fragmented HeLa S3 cellular RNA (20 ng starting material after ribodepletion). Library products for cellular RNAs that were fragmented to 70-90 nt run at 190-210 bp. B. Plasma RNA (0.5 ng of starting material). Library products run at 140-240 bp, with the peak at ~200 bp corresponding to library products from tRNAs. Residual adapter dimers (AD), which are present at low concentrations in the final libraries, run at ~120 bp for R1R-R2R, and slightly larger with the UMI R1R adapter or Unique Dual Index (UDI) PCR primer. Peaks smaller than 100 bp correspond to unused PCR and adapter oligonucleotides (P+A). The lower (35 bp) and upper (10,380 bp) markers are internal markers provided with the High Sensitivity DNA kit. DNA lengths (bp) are determined by the software program based on the ladder provided with the kit.

      
      

      Notes:

      1. If needed, an additional round of the 1.4× AMPure XP beads clean-up can be performed to further deplete levels of adapter dimers. More extensive AMPure XP beads clean-up can result in loss of library products.

      2. The final TGIRT-seq library can be stored at -20°C until submitted for sequencing.

         
         

6. Sequencing and data analysis

   TGIRT-seq libraries can be sequenced on any Illumina sequencing instrument to obtain single-end (SE) or paired-end (PE) reads. Read length and read depth are dependent upon the needs of the experiment. We have used PE75 on a NextSeq 500 with 330 million reads output for analysis of cellular, EV, and plasma RNAs. We have also used PE150 on a NovaSeq with at least 700 million reads output for large projects or samples requiring high-depth analysis.

   Because TGIRT-seq simultaneously profiles coding and non-coding RNAs, read mapping pipelines for TGIRT-seq typically use a sequential mapping strategy with specialized databases to first map reads with rRNA and tRNA sequences encoded at multiple loci, and sncRNAs embedded within introns in protein-coding genes, before mapping the remaining reads to a human genome reference sequence. In addition to end-to-end alignment, a local alignment step is typically included to more efficiently capture reads with non-templated nucleotides added to the 3' end of cDNAs by TGIRT enzymes. Detailed protocols for read processing and mapping, including UMI deconvolution of duplicate reads, and peak calling for the identification of protein-protected mRNA fragments, structured excised intron RNAs, and intron RNA fragments as potential biomarkers in human plasma can be found in Yao et al. (2020) .

   Additional TGIRT-seq read mapping protocols used for different sample types and applications, including statistical tests and details of replicates for different applications, can be found in the following references: plasma RNA (Qin et al., 2016); total cellular RNA, including spike-ins (Nottingham et al., 2016); plasma DNA, including nucleosome positioning and mapping of DNA methylation sites (Wu and Lambowitz, 2017); sncRNAs (Boivin et al., 2018); total cellular RNAs, including detailed analysis and remediation of sequencing biases (Xu et al., 2019).

Notes

1. We have used various commercial kits to isolate RNAs for TGIRT-seq. The commercial kits differ significantly in RNA yield and can bias for different RNA biotypes. We have used the mirVana miRNA Isolation kit (Thermo Fisher Scientific, catalog number: AM1560) with good results for TGIRT-seq of human cellular RNA. For EVs, we obtained good results by using the Total Exosome RNA and Protein Isolation kit (Thermo Fisher Scientific, catalog number: 4478545). For isolating human plasma RNAs, we have tried a number of different extraction reagents and kits, including TRIzol LS Reagent (Thermo Fisher Scientific, catalog number: 10296010) and the Direct-zol RNA Miniprep (Zymo Research, catalog number: R2051); QIAamp ccfDNA/RNA Extraction kit (QIAGEN, catalog number: 55184); and miRNeasy Serum/Plasma Advanced kit (QIAGEN, catalog number: 217204). Each kit has advantages and disadvantages for input amount, ease of use, and RNA yield. In our hands, the QIAamp ccfDNA/RNA kit was more efficient for extracting DNA than RNA (Yao et al., 2020).

2. TGIRT enzymes can template switch from the R2 RNA/R2R DNA starter duplex to the 3' end of DNA fragments, and this activity has been used for sequencing single-stranded DNA for analysis of nucleosome positioning and mapping of DNA methylation sites (Wu and Lambowitz, 2017). For RNA-seq, it is important to remove as much DNA as possible to minimize DNA reads. We have used Turbo DNase (Thermo Fisher Scientific, catalog number: AM2238) and a combination of DNase I (Zymo Research, catalog number: E1010) and Exonuclease I (Lucigen, catalog number: X40520K) (Yao et al., 2020). We clean up DNase-treated RNAs using an RNA Clean & Concentrator-5 kit (Zymo Research, catalog number: R1013) with a modified 8× V/V (ethanol/input RNA) protocol.

3. Cellular RNAs are rRNA-depleted to minimize reads mapping to rRNAs. As rRNA removal procedures are not 100% specific for rRNAs, different rRNA depletion kits can also affect reads corresponding to other RNA biotypes, including RNAs of interest. Thus, the same rRNA depletion kit should be used for all samples in a study. The Illumina Ribo-Zero Plus rRNA Depletion kit (Illumina, catalog number: 20040526) with the manufacturer's protocol gives satisfactory results for TGIRT-seq [5-10% rRNA reads in the final library, although in our hands it is not as effective at removing rRNA as the original (now discontinued) version of that kit (RiboZero Gold, <1-2% rRNA reads)].

4. For chemical fragmentation of cellular RNAs, we use the NEBNext Magnesium RNA Fragmentation Module (New England Biolabs, catalog number: E6150S). We typically use 50 ng rRNA-depleted total cellular RNA as input and heat at 94°C for 5-7 min for fragmentation. The RNA input and fragmentation time should be optimized by the researcher according to the RNAs of interest and desired read length for Illumina sequencing. After fragmentation, we clean up the RNA by using an RNA Clean & Concentrator-5 kit (Zymo Research, catalog number: R1013) with a modified 8× V/V (ethanol/input RNA) protocol. Fragmented cellular RNAs generated with divalent cations should be treated with T4 polynucleotide kinase with 3’-phosphatase activity to remove 3' phosphates and 2’,3’-cyclic phosphates, which impede TGIRT template-switching (Mohr et al., 2013; Nottingham et al., 2016). This inhibition is less pronounced at 200 mM than at 450 mM NaCl (Lentzsch et al., 2019; Yao et al., 2020).

5. The high salt concentration (450 mM NaCl) used in initial TGIRT-seq protocols to suppress multiple end-to-end template switches by the TGIRT enzyme also decreases the efficiency of the initial template-switching reaction from the R2 RNA/R2R DNA Starter Duplex (Lentzsch et al., 2019). Multiple template switches are particularly problematic for miRNA sequencing. The updated protocol using 200 mM NaCl to increase the efficiency of initial template-switching reaction from the R2 RNA/R2R DNA starter duplex gave comprehensive libraries of coding and non-coding RNAs in human plasma with acceptable levels of fusion reads (0.5-4%), which include multiple template switches (Yao et al., 2020).

6. With low RNA inputs (e.g., for human plasma and EV RNAs), Bioanalyzer traces of TGIRT-seq libraries may show an adapter dimer peak but no software-called library product peaks. In those cases, 1 μl of the library can be amplified with another 12 cycles of PCR to detect library products. If library products can be detected after this further PCR amplification, the original library without further amplification should be okay for sequencing. After the additional amplification, adapter dimer levels will be even higher (as the short products amplify preferentially), but the user should expect to observe desired products of the predicted sizes. If library products are still not detected, it likely means that library preparation failed due to an RNA input problem (quality or quantity).

7. A lack of both a product peak and an adapter dimer peak (120-125 bp) in Bioanalyzer traces of the final TGIRT-seq library indicates that the ligation reaction was unsuccessful, most likely due to failure of the R1R adenylation step or ligation.

Acknowledgments

The development of TGIRT-seq methods has been supported by NIH grant R35 GM136216 and Welch Foundation Grant F-1607. This protocol has been adapted from Yao et al. (2020).

Competing interests

Thermostable group II intron reverse transcriptase enzymes and methods for their use are the subject of patents and patent applications that have been licensed by the University of Texas and East Tennessee State University to InGex, LLC. A.M.L, some former and present members of the Lambowitz laboratory, and the University of Texas are minority equity holders in InGex, LLC, and receive royalty payments from the sale of TGIRT enzymes and kits employing TGIRT template-switching activity and from the sublicensing of intellectual property to other companies.

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