Global Run-On sequencing to measure nascent transcription in C. elegans

Summary Global Run-On sequencing (GRO-seq) is one of the most sensitive techniques to detect nascent transcription from RNA polymerase (Pol) at a genome-wide level. The protocol incorporates labeled ribonucleotides into nascent RNAs from Pol I, II, and III. We have adapted the GRO-seq protocol to the nematode Caenorhabditis elegans to measure transcription from embryos and adult worms. Here, we provide a detailed overview of the protocol highlighting the critical steps for generating successful libraries. For complete details on the use and execution of this protocol, please refer to Quarato et al. (2021).


SUMMARY
Global Run-On sequencing (GRO-seq) is one of the most sensitive techniques to detect nascent transcription from RNA polymerase (Pol) at a genome-wide level. The protocol incorporates labeled ribonucleotides into nascent RNAs from Pol I, II, and III. We have adapted the GRO-seq protocol to the nematode Caenorhabditis elegans to measure transcription from embryos and adult worms. Here, we provide a detailed overview of the protocol highlighting the critical steps for generating successful libraries. For complete details on the use and execution of this protocol, please refer to Quarato et al. (2021).

BEFORE YOU BEGIN
Overview of the protocol GRO-seq is a powerful technique to map RNA polymerase transcription at the genome-wide level. The assay has been initially developed for human cells (Core et al., 2008) and has been updated and adapted to work in fission yeasts, Drosophila melanogaster, and plant cells (Mahat et al., 2016). Here we describe the specific steps for producing GRO-seq libraries from Caenorhabditis elegans worms and embryos. We have used this protocol to generate GRO-seq libraries from as low as 1,000 worms or 40,000 embryos (Barucci et al., 2020;Quarato et al., 2021;Singh et al., 2021).
The protocol can be divided into three main steps: 1. Nuclei isolation and nuclear run-on with biotinylated-UTP 2. Enrichment of biotin-labeled nascent RNAs and library preparation 3. Library amplification and sequencing Collection of synchronized worms and early embryos To grow synchronous populations of worms, collect embryos from gravid adults by bleaching and allow hatching for 12-20 h at 20 C to obtain synchronized L1 larvae. Seed 40,000 L1 larvae onto one 150 mm NGM plate and harvest worms at the desired developmental stage. Perform three washes with M9 buffer, remove the supernatant, and flash-freeze in dry ice. Worms can be stored at À80 C for up to a year.
To collect populations of early embryos, grow four 150 mm NGM plates of synchronous worms until adulthood. Carefully monitor worms using a stereomicroscope and perform bleaching shortly after they start to produce the first embryos. Immediately after bleaching, perform three washes with a cold M9 buffer, remove the supernatant and immediately freeze in dry ice to block cell divisions. Embryos can be stored at À80 C for up to a year.

MATERIALS AND EQUIPMENT
Note: we suggest purchasing molecular biology grade reagents for the preparation of the following buffers. However, it is possible to prepare in the lab most of the solutions needed. If so, solutions should be prepared with nuclease-free water and filter-sterilized with a 0.22 mm filter.

STEP-BY-STEP METHOD DETAILS
CRITICAL: all steps of the protocol are performed at 4 C unless stated differently. Pre-chill all reagents and equipment before use. Nuclease-free reagents need to be used in all steps of the protocol.

Nuclei isolation
Timing: 1 h The following steps describe the procedure to isolate intact nuclei from worms or embryos. The protocol has been tested with 1,000 to 40,000 worms and 40,000 to 300,000 early embryos.
1. Resuspend worms or embryos in 1.5 mL of cold Nuclei extraction buffer (NEB) and transfer to a pre-chilled stainless steel tissue grinder on ice. 2. Lyse worms to release nuclei by applying 40 dounce strokes.
CRITICAL: take a small aliquot of the lysate (10-20 mL) and check that the worms are lysed using a stereomicroscope. Intact bodies and embryos should be absent. If intact corpses are still present, increase the number of dounce strokes.
3. Transfer the lysate to a pre-chilled 1.5 mL microcentrifuge tube.

Reagent Final concentration Amount
Triton X-100 (10%) 0.5% 2.5 mL ddH 2 O n/a 25 mL Total n/a 50 mL Buffer can be stored at 4 C for up to 3 months.

Reagent Final concentration Amount
NaCl (

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Note: Multiple samples can be processed simultaneously. If so, keep lysate from step 3 on ice while lysing the other samples. In this case, wash the metal dounce well between each sample with milliQ water. After processing all the samples, proceed immediately to step 4. 4. Remove debris by centrifuging at 1003g for 4 min at 4 C. 5. Transfer the supernatant to a new 1.5 mL low binding microcentrifuge tube.
CRITICAL: carefully collect the supernatant without disturbing the debris pellet. If necessary, repeat steps 4 and 5 until lysate is clear from debris. A residual 50-100 mL can be left in the tube to avoid debris carryover.
CRITICAL: wash nuclei by pipetting up and down several times using a wide-bore tip.
Gently pipette nuclei to preserve their integrity.
8. Pellet nuclei by centrifuging at 1,0003g for 4 min at 4 C. 9. Repeat steps 7 and 8 three more times for a total of 4 washes with NEB. 10. Wash nuclei in 1 mL Freezing buffer NO-Glycerol.
CRITICAL: wash nuclei by pipetting up and down several times using a wide-bore tip.
Gently pipette nuclei to preserve their integrity.
11. Pellet nuclei by centrifuging at 1,0003g for 4 min at 4 C and discard the supernatant. 12. Resuspend nuclei in 100 mL Freezing buffer.
Pause point: nuclei in the Freezing buffer can be stored at À80 C for up to 1 year.

Nuclear run-on (NRO) reaction
Timing: 15 min NRO is performed on isolated nuclei in the presence of Bio-11-UTP. Sarkosyl is added to prevent de novo assembly of the pre-initiation complex and avoid new transcriptional initiation. A negative NRO control reaction can be added at this step. In the negative NRO reaction, UTP is used instead of Biotinylated-UTP. If you are performing the experiment for the first time, the addition of negative control is strongly recommended.
13. Prepare the NRO 2x mix (the recipe is listed in the ''Materials and equipment'' section), and preheat it at 30 C before use. 14. Dispense 80 mL of the NRO 23 mix in a new low binding microcentrifuge tube. 15. Add 20 mL of Bio-11-UTP 10 mM to the NRO 23 mix (final Bio-11-UTP concentration: 1 mM) and gently mix.
CRITICAL: if running a negative NRO reaction, replace Bio-11-UTP with 0.5 mL of UTP 100 mM and 19.5 mL of nuclease-free water.
16. Add 100 mL of lysed nuclei from the previous step 12. Gently mix the reaction well using a wide bore tip. 17. Incubate the reaction for 3 min at 30 C. 18. Transfer the tubes on ice and add 50 mL ice-cold nuclease-free water and 750 mL TRIzol LS reagent and mix well to stop the reaction. 19. Incubate nuclei in TRIzol for 5 min at room temperature (20 C-25 C) to permit complete dissociation of the nucleoprotein complexes.
CRITICAL: TRIzol has an acute oral and dermal toxicity. Always handle in a chemical hood.
Contact with eyes and skin should be avoided.
Pause point: Nuclei in TRIzol can be stored at À80 C for up to a week (we have not tested longer storage).
CRITICAL: BCP is toxic upon inhalation. Always handle in a chemical hood.
21. Incubate at RT for 15 min. 22. Centrifuge at 12,0003g for 15 min at 4 C, then transfer the aqueous phase (upper phase) containing the RNA to a new low binding microcentrifuge tube.
Note: Avoid transferring any of the interphase or organic layer into the pipette when removing the aqueous phase. The tube can be angled at 45 to facilitate the process. Approximately 450 mL of aqueous phase can be collected at this step.
23. Add 2 mL of GlycoBlue and mix it well by vortexing for 5-10 s. 24. Add 500 mL of isopropanol and mix it vigorously by vortexing for 10-15 s. 25. Incubate at RT for 15 min to precipitate the RNA. 26. Centrifuge at 12,0003g for 15 min at 4 C to pellet the RNA. 27. Remove the supernatant and add 1 mL of 75 % Ethanol to the RNA pellet.
Note: After centrifugation, a blue pellet should be visible at the bottom of the tube.
CRITICAL: Carefully remove the supernatant without disturbing the RNA pellet.
29. Remove the supernatant and air dry the pellet for 1-2 min.
Note: to completely remove the supernatant without disturbing the pellet you can use the following procedure: a. Remove the supernatant using a p1000 micropipette. b. Briefly centrifuge to collect all residual supernatant at the bottom of the tube. At this point, the pellet should be on the side of the tube. c. Using a p200 micropipette, gently push at the bottom-center of the tube and remove all residual ethanol. Always check that the pellet is not entering the tip. d. Briefly air dry the pellet.
CRITICAL: do not over-dry the RNA pellet as this would make it difficult to resuspend it.

RNA fragmentation
Timing: 8 min 30. Resuspend the RNA pellet from the previous step 29 in 8 mL water.
Pause point: RNA can be stored at À80 C for up to a week.
Note: The analysis of RNA quality/integrity is not required at this step. This is because degradation fragments cannot be cloned with the library preparation strategy described in this protocol. Only RNA molecules with 3 0 -hydroxyl biotinylated ends will be suitable for cloning. In addition, the RNA quality control methods will primarily detect mature mRNAs and not Polymerase-bound nascent RNAs, which are much less abundant. 31. Add 2 mL of 53 reverse transcriptase buffer (provided with SuperScriptä IV Reverse Transcriptase) and mix it well by pipetting or vortexing. Spin down the samples using a benchtop centrifuge.
Note: RNA fragmentation can be performed using different buffers, including commercially available RNA fragmentation reagents, based on MgCl 2 or ZnCl 2 . If using a different buffer, check that the final library size is correct and adjust the fragmentation time accordingly.
32. Incubate the samples at 95 C for 7 min.
Note: incubation can be performed in a ThermoMixer equipped with Thermo Top. Alternatively, the sample can be transferred to a PCR tube and incubated in a thermocycler with the lid temperature set at 105 C. During the fragmentation time, it is possible to start preparing the Dynabeads magnetic beads to isolate biotinylated molecules.
33. Immediately block fragmentation by shifting the samples on ice. 34. Add 50 mL of ice-cold nuclease-free water and keep on ice for at least 1 min before proceeding to the next step.

Preparation of dynabeads MyOne streptavidin C1
Timing: 10 min Dynabeads are washed following the manufacturer's instructions before binding to biotinylated RNA.
CRITICAL: This step is crucial since the beads are not supplied in RNase-free solutions.
Note: all the washing steps are performed at room temperature (20 C-25 C) unless stated differently.
35. Resuspend the Dynabeadsä magne/tic beads in the vial (i.e., vortex for >30 s, or tilt and rotate for 5 min). 36. Transfer 30 mL of Dynabeadsä magnetic beads per sample to a low binding microcentrifuge tube.
Note: 30 mL of beads are sufficient for a sample of 1,000 to 40,000 adult worms or 40,000 to 300,000 embryos. If performing the experiment with a different number of worms/embryos, the volume of beads should be scaled accordingly. Note: The washed beads can be prepared in advance and stored at 4 C for 1 day.

st enrichment of biotinylated RNA molecules
Timing: 50 min Biotinylated nascent RNAs are enriched by binding to Dynabeadsä magnetic beads. After binding, the beads coated with the biotinylated molecules are washed using high salt buffers to efficiently remove non-biotinylated molecules.
45. Add 60 mL of washed beads from previous step 44 to 60 mL of fragmented RNA from previous step 34. 46. Incubate for 30 min at 25 C with 1200 rpm agitation (15'' ON 45'' OFF) in a ThermoMixer. 47. Briefly spin the tubes using a benchtop centrifuge and place on a magnetic rack for 1 min or until the solution appears clear. 48. Remove the supernatant and add 1 mL of High salt buffer. Incubate on rotation for 5 min at 4 C. 49. Briefly spin the tubes using a benchtop centrifuge and place on a magnet for 1 min or until the solution appears clear. 50. Remove the supernatant and add 1 mL of Medium salt buffer. Incubate on rotation for 5 min at 4 C. 51. Briefly spin the tubes using a benchtop centrifuge and place on a magnet for 1 min or until the solution appears clear. 52. Remove the supernatant and add 1 mL of Low salt buffer. Incubate on rotation for 5 min at 4 C. 53. Briefly spin the tubes using a benchtop centrifuge and place on a magnet for 1 min or until the solution appears clear. 54. Remove the supernatant, add 1mL TRI Reagent solution, and proceed to RNA isolation as previously described (Refer to ''RNA isolation using TRIzol'', steps 20-29).
Pause point: Beads in TRIzol can be stored at À80 C for up to a week.

RNA 5 0 phosphorylation
Timing: 35 min The RNA fragmentation step produces fragmented RNA molecules with 3 0 -phosphate and 5 0 -hydroxyl termini, not suitable for the adapter ligation steps, which require a 3 0 -hydroxyl and a 5 0 -phosphate ll OPEN ACCESS terminus. Therefore, with the following steps, 5 0 -hydroxyl ends are phosphorylated using the Polynucleotide kinase.
Note: Since incorporating Bio-11-UTP stall RNA polymerases, all nascent RNAs should terminate with a 3 0 -hydroxyl biotinylated end. Therefore, RNA molecules terminating with a 3 0 -phosphate end, result from the fragmentation steps and represent the background for this protocol. For this reason, 3 0 -phosphate ends do not need to be repaired to allow ligation of the adapter.
Pause point: RNA can be stored at À80 C for up to a week.
56. Setup RNA 5 0 phosphorylation reaction in 100 mL final volume by adding to the RNA from the previous step: a. 10 mL 103 Polynucleotide Kinase Buffer( b. 1 mL 100 mM ATP c. 0.5 mL 40 U/mL RNase inhibitor d. 2mL 100 U/mL T4 Polynucleotide Kinase e. 66.5 mL water. 57. Incubate the reaction at 37 C for 30 min. 58. Add 200 mL of ice-cold nuclease-free water, keep the sample on ice and proceed to the next step.
CRITICAL: do not vortex the tube to avoid disturbing the Phase Lock gel.
CRITICAL: Phenol:Chloroform is toxic upon inhalation. Always handle in a chemical hood.
Contact with eyes and skin should be avoided.
62. Centrifuge at 12,0003g for 5 min at room temperature (20 C-25 C) and transfer the aqueous phase containing the RNA (upper phase) to a fresh low binding microcentrifuge tube. 63. Add 30 mL of 3 M sodium acetate and 2 mL of GlycoBlue and vortex for 5-10 s. 64. Add 900 mL ethanol and vortex vigorously for 10-15 s. 65. Precipitate the RNA by incubating for 1h at À20 C.
Pause point: RNA can be precipitated overnight or over-weekend.
66. Centrifuge to pellet the RNA at 12,0003g for 30 min at 4 C. 67. Remove the supernatant and add 1 mL of 75 % Ethanol to the RNA pellet.   CRITICAL: Over-drying the sample may result in a lower recovery.
83. Remove tubes from the rack and elute purified RNA from the beads by adding 61 mL water.
CRITICAL: manually resuspend the beads by pipetting up and down several times. Expel the elution buffer down the side of the tube to ensure the entire bead mass comes into contact with the buffer. Incubate for 30 s before proceeding to the next step.
84. Place the tubes onto a magnetic tube rack for 1 min to separate the beads from the solution.
Then, slowly collect 60 mL RNA solution and transfer to a fresh tube.
CRITICAL: slowly collect the solution while keeping the tubes onto the magnetic rack to avoid beads' carryover. 1 mL of excess water is added at step 83 is added to avoid beads' carryover.
Pause point: RNA can be stored at À80 C for up to a week. 93. Resuspend RNA after TRIzol purification in 11 mL of water. 94. Perform reverse transcription reaction in a final volume of 20 mL by adding to RNA from the previous step: a. 1 mL SR_RT primer 50 mM b. 1 mL dNTP mix 10 mM c. 4 mL SSIV Buffer 53 d. 1 mL DTT 100 mM e. 1 mL RNase inhibitor f. 1 mL SuperScript IV reverse transcriptase 95. Mix well and incubate the reaction at 50 C for 1 h in a thermal cycler with the lid temperature set at 85 C. 96. Inactivate the reaction by heating the sample at 80 C for 10 min. 97. Keep the sample at 4 C until ready to proceed to the next step.
Pause point: reverse-transcribed cDNA can be stored at À20 C for up to a month.

OPEN ACCESS
After reverse transcription, libraries are amplified by PCR using a universal primer and a barcoded primer to allow sample multiplexing. Primer sequences are listed in Table 1.
The number of amplification cycles required to amplify the libraries is influenced by many factors and needs to be determined experimentally for each sample (for example, very early embryos have lower transcription levels than late embryos). Therefore, we suggest performing a test PCR amplification of the libraries using 1 / 4 of the reverse transcription reaction.
CRITICAL: The number of PCR cycles should be minimized to avoid overamplification, which increases PCR artifacts. Please refer to Figure 1 for an example of a correctly amplified library.
Note: if multiple samples need to be sequenced together, use a different SR barcode primer per sample. The sequence of the SR barcode primers is available in Table 1. 98. Setup PCR reaction in a final volume of 50 mL by combining: a. 17 mL water b. 5 mL reverse-transcribed cDNA library c. 1.5 mL 10 mM SR universal primer d. 1.5 mL 10 mM SR barcode primer 10 e. 25 mL NEBNextâ Ultraä II Q5â Master Mix 23 99. Place the tube on a thermocycler with the heated lid set to 105 C and perform PCR amplification using the following PCR cycling conditions: Note: We generally perform 20 PCR cycles for the test PCR for both adults and embryos.
Purification of the PCR reaction using agencourt AMPure XP beads

Timing: 30 min
AMPure SPRI beads are used to remove salts, dNTP, primers, and primer dimers from the PCR reaction. 1.8 volumes of AMPure beads are used to recover fragments > 100 bp (expected size of amplicons 140-350 bp).
Alternatives: equivalent SPRI beads can be used at this step instead of Agencourt AMPure XP beads. If different beads are used, the beads/sample ratio might vary. Refer to manufacturer guidelines to select the proper beads/sample ratio to purify fragments > 100 bp.

Figure 1. Tapestation analysis showing a proper library preparation result
Example electropherograms from Tapestation analysis of GRO-seq libraries using 1,000 worms (A), 100,000 embryos (B), and 100,000 embryos after a negative NRO control reaction using UTP instead of Bio-11-UTP (C). No residual primers (< 100 bp) nor overamplification products (> 600 bp) are visible. The peak height is in the same range as the lower and upper markers for Figures (A and B). Number of PCR cycles used to amplify each library is indicated in the figure.

PCR cycling conditions
Steps 101. Mix well by pipetting up and down at least 15 times or by gentle vortexing and incubate for 5 min at RT. 102. Briefly spin using a benchtop centrifuge and place the tube onto a magnetic tube rack for 5 min to separate the beads from the solution. 103. Slowly pipette the cleared solution from the tube and discard. 104. While on the magnetic rack, dispense 200 mL of 75% ethanol into the tube, incubate for 30 s and remove the Ethanol. 105. Repeat the previous step for a total of 2 washes. 106. Remove the tube from the magnetic rack and briefly spin down residual ethanol using a benchtop centrifuge. 107. Place the tube onto a magnetic tube rack and remove residual ethanol. 108. Let the beads air fry for 5 min.
CRITICAL: Over-drying the sample may result in a lower recovery.
109. Remove the tube from the rack and elute purified DNA from the beads by adding 18 mL water.
CRITICAL: manually resuspend the beads by pipetting up and down several times. Expel the elution buffer down the side of the tube to ensure the entire bead mass comes into contact with the buffer. Incubate for 30 s before proceeding to the next step.
110. Place the tubes onto a magnetic tube rack for 1 min to separate the beads from the solution.
Then, slowly collect 17 mL of cleaned DNA libraries and transfer them to a fresh tube.
CRITICAL: slowly pipette the solution while keeping the tubes onto the magnetic rack to avoid beads' carryover 1 mL of excess water is added at step 110 to prevent beads carryover.

Quality control of purified DNA libraries and full-scale PCR amplification
Timing: 10 min Purified DNA libraries are run on an Agilent Tapestation instrument using the High Sensitivity D1000 reagents to check for library size, purity, and amount.
111. Run 2 mL purified DNA libraries on an Agilent Tapestation instrument using the High Sensitivity D1000 reagents following manufacturer conditions. 112. Examine the result of the Tapestation run and determine the right PCR cycles for the full-scale PCR amplification. An example of Tapestation results for both worms and embryos can be found in the expected outcomes section and Figure 1.
CRITICAL: Adjust the PCR cycles of the final PCR amplification, calculating that each library should be in the same amount range of the upper and lower markers. Check for the presence of over-amplification products (high molecular size products) and reduce the number of PCR cycles to eliminate them (See expected outcomes, troubleshooting section, and Figure 3). CRITICAL: This step is crucial to assess the amount of each DNA library, the amplification status, and the absence of residual PCR primers before quantifying and sequencing. An example of TapeStation results for both worms and embryos can be found in the section expected outcomes and Figure 1.

Quantification of DNA libraries and pooling for next-generation sequencing
Timing: 20 min In the following steps, libraries are precisely quantified and combined in an equimolar ratio based on the size distribution and the concentration.

LIMITATIONS
Here, we presented a protocol to perform GRO-seq on as low as 1,000 L4/adult worms (around 2,000,000 nuclei) or 40,000 early embryos (around 800,000 nuclei). The input required for this protocol is much lower than the first reported GRO-seq in C. elegans, which required 100,000 worms (Cecere et al., 2014). Nonetheless, this minimum number of worms or embryos might still be difficult to achieve for specific samples, such as mutant strains with fertility defects. The main limitation of the protocol is the nuclei extraction procedure which has an estimated efficiency of 50%. Therefore, improvements in this step might help decrease the input material required to run GRO-seq in the future.
Another limitation is the high cost of the method. Indeed, reagents such as biotinylated nucleotides, streptavidin-conjugated beads, and ligases drastically influence the cost per sample. Home-production of the required enzymes and order of large batches of biotinylated nucleotides helps reduce the cost per sample of this protocol.
Ultimately, to understand the advantages and the limitations of the general GRO-seq procedure, we suggest referring to (Mahat et al., 2016).

Problem 1
No library product or > 25 PCR amplification cycles are required to obtain the libraries (step 112).
Possible causes: Figure 2. Genome browser example of a GRO-seq library compared to RNA-seq libraries A genomic view of two protein-coding genes showing normalized GRO-seq (orange) or RNA-seq (light purple) reads from early embryos or adults. oma-1 (left) is highly expressed in adult worms and is among the embryo's most abundant maternally inherited mRNAs. However, oma-1 is not transcribed in early embryos. vet-6 (right) is not expressed in adult worms, is not maternally inherited, and is among the earliest transcribed genes in embryos.

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Poor nuclei preparation quality.
Problems in the NRO reaction.
Low recovery in the SPRI purification steps.

Potential solution
Potential solutions are listed below: Stain nuclei with DAPI and check for their integrity by fluorescence microscopy. If nuclei are not intact, reduce the number of dounce strokes at step 2.
Check for precipitates in the NRO 23 mix. If present, prepare the solution again.
Always work on ice and use RNase-free reagents at all steps.
Be careful not to over-dry the beads after purification. Dry beads are difficult to resuspend and may result in low recovery of RNA or DNA.

Potential solution
If primers are present (< 100 bp), perform a new round of library size selection using AMPure SPRI beads and perform a new Tapestation analysis.
Note: We do not recommend performing gel purification to remove primer-dimers as this would result in sample loss. In our experience, an additional round of SPRI beads purification is sufficient to remove unused primers or primer-dimers and to obtain libraries of high quality.

Problem 3
Libraries are overamplified and show peaks corresponding to high molecular weight products (> 600 bp) (step 112).

Potential solution
Reduce PCR amplification by 2-4 cycles and perform a new Tapestation analysis. Figure 3 shows an example of the same library amplified with different PCR cycles.

Problem 4
Streptavidin magnetic beads adhere to the tube wall after washing with solution A (step 42).

Potential solution
The addition of 0.02% tween to Solution A can help to prevent this issue. Also, the use of low binding microcentrifuge tubes is suggested.
Furthermore, we have noticed that solution A might form precipitates few days after preparation. Therefore, we suggest preparing a new batch of solution A for each experiment.

Problem 5
Small or no pellet recovered after TRIzol or Phenol:Chloroform purification (steps 26 or 66).

Potential solution
The absence of pellet after RNA precipitation might be due to incomplete mixing of the aqueous phase containing the RNA with GlycoBlue and Isopropanol/Ethanol (steps 23-24 for TRIzol purification and 64-65 for Phenol:Chloroform purification).
Mix the sample again by vortexing at maximum speed for 15 s and repeat the precipitation and centrifugation steps.
If the pellet is still not visible, add 2 mL of GlycoBlue, mix thoroughly, and repeat the precipitation and centrifugation steps by increasing the centrifugation speed to 16,0003g.

RESOURCE AVAILABILITY
Lead contact Further information and requests for resources and reagents should be directed to and fulfilled by the lead contact, Germano Cecere (germano.cecere@pasteur.fr).