Tissue NET-seq (native elongating transcript sequencing)

Place the fresh liver tissue in ice-cold PBS, cut into smaller pieces and homogenize in 3 ml of nuclei isolation buffer using a Dounce tissue homogenizer (Wheaton). After complete homogenization, transfer the sample to a Falcon tube. Add 3 ml of nuclei isolation buffer to the remaining pieces in the tissue homogenizer, homogenize further and transfer the remnants also to the tube. Following an incubation on ice for 5 min, pass the samples through a 70-μm cell strainer and collect the nuclei by centrifuging at 500 x g for 20 min.

Resuspend the nuclei pellet in 4 ml of nuclei isolation buffer. After incubation on ice for 5 min, collect the nuclei by centrifuging at 500 x g for 5 min. To remove cytoplasmic remnants, wash the nuclei pellet with 1600 μl nuclei wash buffer and centrifuge at 1,150 x g for 5 min. Repeat the washing with 800 μl nuclei wash buffer. Then, gently resuspend the pellet in 200 µl of glycerol buffer using a cut 1,000-µl tip and transfer the suspension to a new 1.5-ml RNase-free microcentrifuge tube. To lyse nuclei, add 400 µl of nuclei lysis buffer, mix samples by pulsed vortexing and incubate on ice for 20 min. After assessing nuclei lysis of a small aliquot under a light microscope, centrifuge the samples at 18,500 x g for 2 min. Completely remove the supernatant (nucleoplasmic fraction) and resuspend the chromatin pellet in 50 µl chromatin resuspension solution.

Using a cut P1000 pipette tip, add 200 µl of CLB to cells and mix by pipetting the sample up and down ten times. Transfer sample to an RNase-free 1.5 ml microcentrifuge tube using a cut 1,000 µl pipette tip.

Incubate the cell lysate on ice for 5 min.

1. Add 700 μl of QIAzol lysis reagent (part of miRNeasy mini kit, Qiagen) to the resuspended chromatin

2. Mix thoroughly by slowly pipetting up and down using a 1-ml syringe with a 22G needle. Alternatively, the chromatin pellet can also be solubilized by gentle vortexing. Mix very carefully until the solution is homogeneous. Mix it slowly to avoid spilling the sample. (at this point the sample can be stored at −80 °C for months).

3. Prepare RNA using the miRNeasy mini kit according to the manufacturer’s instructions, including the optional on-column DNase treatment using the RNase-free DNase set (Qiagen).

4. Elute the nascent RNA in 25 µl RNase-free H₂O. Assess the quantity and quality of the prepared RNA using NanoDrop 2000 (Thermo Scientific). RNA yield from one whole liver typically ranged from 10 to 56 µg. The absorbance A260/A280 ratios were around 2.1.

During incubation period, pipette 500 µl of SB to an RNase-free 1.5 ml microcentrifuge tube.

1. Spray down all work surfaces, pipettes, etc. with RNase away

2. Denature the RNA sample (from the cellular fractionation, section 1.3.3) and 5 µl of the oGAB11 control (10 µM; Table 1 in the Materials section) for 2 min at 80 °C in a Thermomixer. Place on ice.

3. Prepare linker ligation mix for each RNA sample and for oGAB11 in 0.2-ml RNase-free PCR tubes (see Materials section 1.4.1, Linker ligation, step 4). Mix until reaction is homogeneous, poor mixing will negatively affect the ligation efficiency.

4. Add 1 µl Truncated T4 RNA ligase 2 (200U) to each RNA sample as well as the oGAB11 control.

5. Incubate the ligation samples overnight for 10 - 16 hours at 16 °C in a thermal cycler (Note 31 in the Guidelines section).

6. Place on ice and add 0.7 µl of EDTA (0.5 M) to each ligation sample to stop the ligation reaction.

A	B	C	D
Component	Amount per reaction (µl)	Final
RNA sample	oGAB11 control
PEG8000 (50% v/v)	8.0	8.0	20% (v/v)
DMSO	2.0	2.0	10 % (v/v)
T4 RNA ligase buffer (10x)	2.0	2.0	1x
Barcode DNA linker (1 µg)	1.0	1.0
RNA sample (1 µg)	6.0	-
oGAB11 (10 µM)	-	1.0	0.5 µM
RNase-free H2O	-	5.0
Truncated T4 RNA ligase 2	1.0	1.0	200 U

After incubation period, use a cut 1,000 µl pipette tip to layer the cell lysate sample onto the SB (see Note 27).

Prior to RNA fragmentation, precipitate the RNA to remove PEG, which can affect the fragmentation reaction.

1. To each sample, add 60 µl of 3 M sodium acetate, 2 µl GlycoBlue (15 mg/ml) and 600 µl of 100 % ethanol.

2. ncubate the samples at -80°C for 45 min. (precipitates can be stored at -80 °C overnight

3. Centrifuge the samples for 30 min at full speed.

4. Wash the samples twice with 500 µl of each 100 % ethanol and 70 % ethanol.

5. Air-dry the pellets for 5 min and resuspend in 20 µl sterile water (10 µl for oGAB11 ligation control).

Centrifuge layered lysate at 16,000g for 10 min at 4 °C to collect nuclei.

Following precipitation, add 20 µl of 2× alkaline fragmentation solution to each sample and mix. Please note that Steps 4.3 and 5.1 are not performed for the oGAB11 ligation control.

1. Fragment the RNA at 95 °C in a thermal cycler for 20 min. The fragmentation time needs to be adjusted whenever a new batch of alkaline fragmentation solution is applied. An over-fragmentation or under-fragmentation of the nascent RNA pool can lead to systematic biases. In a typical experiment RNA is fragmented between 10 and 40 min at 95 °C. The optimal fragmentation time is when most RNA molecules are in the required size range, usually between 35 and 100 nt.

2. Following fragmentation, precipitate the RNA and the oGAB11 ligation control as described above (steps 4.1-4.3).

3. Add 20 µl of 2× TBE-urea denaturing sample buffer to each RNA sample and mix it.

4. Prepare the RNA control ladder and the oGAB11 control. Add 1.0 µl 0.5 μl of RNA control ladder or 1 μl oGAB11 to 9 µl of RNase-free H₂O. Add 10 µl of 2× TBU denaturing sample buffer to each control sample and mix it.

5. Denature the RNA sample and RNA control samples (oGAB11 ligation control, ladder and oGAB11) for 2 min at 80°C. Cool the samples on ice for 3 min.

6. Pre-run a 15% (wt/vol) polyacrylamide TBE-urea gel at 200 V for 15 min in 1× TBE.

7. Separate the fragmented RNA samples and the RNA control samples (including the oGAB11 ligation control) by PAGE at 200 V for 65 min.

8. Stain the gel in 50 ml of gel staining solution for 5 min at room temperature on a shaker. Protect the gel from light during staining by the use of a black gel box.

9. Visualize the fragmented RNA and the oGAB11 ligation control under blue light. For the fragmented RNA, excise the region between 35 (in the middle of the dye front) and 100 nt. For the oGAB11 ligation control, excise the narrow band at ~55 nt.

10. Extract the RNA from the gel slice by rapid gel extraction:

a. Pierce the bottom of a 0.5-ml RNase-free microcentrifuge tube with a 23G needle.

b. Put the pierced 0.5-ml tube in a 1.5-ml RNase-free microcentrifuge tube.

c. Combine and place the gel slices for each sample into the inner pierced 0.5-ml tube.

d. Centrifuge the mixture at 20,000g for 4 min at room temperature.

e. Add 200 µl RNA recovery buffer (Zymo Research, R1070-1-10) to the excised gel slices.

f. Incubate the sample for 15 min at 70°C in a Thermomixer (1500 rpm).

g. Vortex the mixture for 30 s at a medium intensity setting.

h. Cut the tip off of a 1,000-µl pipette tip and transfer the gel slurry into a Zymo-Spin IV Column (Zymo Research, C1007-50).

i. Freeze for 2 min at -80°C and subsequently thaw by placing at 70°C for 1 min.

j. Centrifuge the mixture at 20,000g for 30 secat room temperature.

k. The expected eluate volume is ~200 µl.

11. Precipitate the RNA by adding 90 µl of 3 M sodium acetate, 2 µl GlycoBlue (15 mg/ml) and 900 µl of 100 % ethanol. Proceed as described above (step 28).

12. Re-suspend the size-selected RNA and the oGAB11 ligation control in 10 µl of pre-cooled RNase-free H₂O.

Pipette off supernatant completely from the nuclei pellet (supernatant contains the cytoplasmic fraction, retain for western blot, described in Box 1)

1. Synthesize cDNA using the SuperScript™ III First-Strand Synthesis System (Thermo Fischer) following the manufacturer’s instructions. In brief, add 0.8 µl dNTPs (10 mM) and 0.5 µl of the reverse primer oLSC007 (10 µM, Table 2) to 10 µl of RNA sample and to the oGAB11 control. After incubating at 80°C in a thermal cycler for 2 min and cooling on ice for 3 min, add 10 µl of cDNA synthesis mix consisting of 2 µl of 10x RT buffer, 4 µl of 25 mM MgCl₂, 2 µl of 0.1 M DTT, 1 µl of RNaseOUT^™ (40 U/µl) and 1 µl of SuperScript^® III RT (200 U/µl) to each sample.

2. Incubate the mixture for 30 min at 48°C in a thermal cycler.

3. Add 1.8 µl of 1 N NaOH, mix well and incubate the reaction for 20 min at 98°C.

4. Neutralize the reaction by adding 1.8 µl of 1 N HCl; mix well and put the reaction on ice.

5. Add 20 µl of 2× TBU denaturing sample buffer to each cDNA sample and the oGAB11 cDNA control, and then mix.

6. Prepare the DNA control ladder. Add 1.0 µl of DNA control ladder to 9 µl of RNase-free H₂O. Add 10 µl of 2× TBU denaturing sample buffer to the ladder and mix.

7. Denature the cDNA sample, oGAB11 cDNA control and DNA control ladder for 3 min at 95°C in a Thermomixer. Cool the samples on ice for 3 min.

8. Pre-run a 10% (wt/vol) polyacrylamide TBE-urea gel at 200 V for 15 min in 1× TBE.

9. Separate the cDNA sample, the oGAB11 cDNA control and the DNA ladder by PAGE at 200 V for 65 min.

10. Stain the gel in 50 ml of gel staining solution for 5 min at room temperature on a shaker. Protect the gel from light during staining by the use of a black gel box.

11. Visualize the gel under blue/UV light and excise the cDNA between 85 and 160 nt.

12. Extract the cDNA from the gel slice by rapid gel extraction (see step 5.10). To 200 µl eluate, add 25 µl of 5 M NaCl and mix.

13. Precipitate the cDNA by adding 2 µl GlycoBlue (15 mg/ml) and 750 µl of 100 % ethanol. Proceed as described above (step 28). Resuspend cDNA and oGAB11 control cDNA in 15 µl of pre-cooled RNase-free H₂O each. The cDNA can be stored indefinitely at −20°C.

Wash nuclei pellet with 800 µl of NWB (either by inverting the tube or pipetting up and down).

1. Prepare circularization mix (Table 4) and store it on ice.

2. Add 4 µl of circularization mix to 15 µl of cDNA sample and the oGAB11 cDNA control sample in a 0.2-ml RNase-free PCR tube, and then mix well.

3. Add 1 µl of CircLigase (100 U/µl) and mix.

A	B	C
Component	Amount per reaction (µl)	Final
CircLigase reaction buffer (10x)	2.0	1x
ATP (1 mM)	1.0	50 µM
MnCl2 (50 mM)	1.0	2.5 mM

4. Incubate the CircLigase reaction for 60 min at 60°C and for 10 min at 80°C in a thermal cycler. Circularized cDNA can be stored indefinitely at −20°C.

Collect cell nuclei by centrifugation at 1,150g for 1 min at 4 °C.

1. For depletion of the 20 most abundant chromatin-associated mature RNAs (see Materials, step 11), prepare one specific depletion reaction per sample in 0.2-ml DNase-free PCR tubes. Please note that the oGAB11 control sample is not subjected to the depletion procedure.

2. Perform subtractive hybridization in a thermal cycler (Table 7).

3. Prepare Dynabeads MyOne streptavidin C1 (10 mg/ml) at room temperature.

a. Re-suspend the beads by gentle vortexing.

b.Transfer 100 µl of beads per depletion reaction to a DNase-free 1.5-ml microcentrifuge tube.

c.Place the tube on a magnetic rack for 1 min and withdraw all of the supernatant from the tube.

d.Remove the tube from the magnetic rack and re-suspend beads in 100 µl of bind/wash buffer.

e.Repeat this washing procedure (steps 3 and 4) two more times.

f.Place the tube on a magnetic rack for 1 min and withdraw the supernatant from the tube.

g.Remove the tube from the magnetic rack and re-suspend the beads in 30 µl of bind/wash buffer.

h.Transfer 25 µl of the re-suspended and washed beads to a new tube.

i.Place the tube in a Thermomixer at 37°C to equilibrate for 15–30 min.

4. Transfer 40 µl of depletion reaction directly from the 0.2-ml PCR tube in the thermal cycler (from Step 71) to the washed and equilibrated beads in the Thermomixer. Immediately mix by pipetting.

Composition of the depletion reactions.

A	B	C
Component	Amount per reaction (µl)	Final
Circularization reaction (from step 7.4)	20
Depletion DNA oligo pool	4.0	1 µM
SSC, 20x	4.0	2x
DNase-free H2O	12.0

Parameters for depletion of highly abundant mature RNAs.

A	B	C
	Temperature	Time
Denature	99°C	90 s
Annealing	99-37°C in 0.1°C steps	1s (per 0.1°C step)
Final annealing	37°C	15 min

5. Incubate the mixture in the Thermomixer for 15 min at 37°C with mixing at 1,000 rpm.

6. Transfer the tubes from the Thermomixer into a magnetic rack and leave them for 1 min. Transfer the supernatant into a new 1.5-ml microcentrifuge tube. The supernatant needs to be transferred carefully. Any remaining magnetic beads in the supernatant will have a negative impact on subsequent steps.

7. Precipitate the oligo-depleted, circularized cDNA by adding 6 µl of 5 M sodium chloride, 2 µl GlycoBlue (15 mg/ml) and 250 µl of 100 % ethanol. Proceed as described above (step 4.1-4.3). Resuspend the cDNA in 10 µl sterile water. DNA can be stored indefinitely at −20°C.

Pipette off supernatant from the nuclei pellet. It is important to remove the supernatant completely in order to remove cytoplasmic mature RNAs (see Note 26).

1. Prepare PCR mix for four pilot PCR amplification reactions for both the cDNA sample and the oGAB11 control cDNA sample (Table 8). Mix well and store it on ice.

2. For each PCR, put 19 µl of PCR master mix in a 0.2-ml RNase-free PCR tube.

3. Add 1 µl of circularized cDNA and mix it well.

4. Perform PCR pilot amplifications (Table 9). Remove one PCR tube for each sample at the end of the extension step after 6, 8, 10 and 12 cycles.

Composition of PCR amplification reactions.

A	B	C
Component	Amount for 4 reactions (µl)	Final
Phusion HF buffer (5x)	15.2	1x
dNTPs (10 mM)	1.5	0.2 mM
Forward primer (Ilumina index primer, 100 µM)	0.4	0.5 µM
oNTI231 (reverse primer, 100 µM)	0.4	0.5 µM
DNase-free H2O	57.6
Phusion DNA polymerase (2 U/µl)	0.9	1.8 U

Parameters for PCR amplification.

A	B	C	D
Cycle number	Denature	Anneal	Extend
1	98°C, 30s
2-14	98°C, 10 s	60°C, 10 s	72°C, 5 s

5. Add 3.4 µl of 6× DNA loading dye to each tube and mix well.

6. Prepare DNA control ladder. Add 1.0 µl of DNA control ladder to 9 µl of DNase-free H₂O. Add 2 µl of 6× DNA loading dye and mix well.

7. Separate the PCR products and the DNA control ladder by TBE gel electrophoresis on an 8% (wt/vol) TBE gel at 180 V for 55 min.

8. Stain the gel in 50 ml of gel staining solution for 5 min at room temperature on a shaker. Protect the gel from light during staining by the use of a black gel box.

9. Visualize the gel under blue/UV light and identify the optimal PCR amplification cycle for each cDNA sequencing library. The NET-seq library runs at ~150 nt. The optimal PCR amplification cycle is characterized by a clear band at ~150 nt and the absence of PCR products at the higher-molecular-weight range.

10. Perform four PCR amplification reactions per sample with the optimal amplification cycle. The oGAB11 control sample is not subjected to amplification. Add 6 µl of 6× DNA loading dye to each sample and mix well. Separate the PCR products by TBE gel electrophoresis on a 4% (wt/vol) low melt agarose gel at 80 V for 2 h.

11. For each sample, excise the band that contains the PCR product from the gel. Excise the broad band at ~150 nt. Avoid contamination from the lower band that runs at ~120 nt, representing PCR product from empty circles. Empty circles are circularized cDNA molecules that arise from unextended RT primers, and hence they do not contain any information about the original nascent RNA.

12. Purify the final NET-seq library using the Nucleospin^™ Gel and PCR Clean-up kit (Macherey-Nagel) according to the manufacturer’s instructions. Elute in 10 µl of Tris-HCl (10 mM, pH 8.0). The DNA sequencing library can be stored indefinitely at −20°C.

Add 200 µl of GB to the nuclei. Use a cut 1,000 µl tip to resuspend the washed nuclei pellet by pipetting up and down.

1. Prepare a 1:5 dilution of the NET-seq library by adding 1 µl of the NET-seq library to 4 µl of Tris-HCl (10 mM, pH 8.0); mix well.

2. Use 1 µl of the diluted NET-seq library for quantification with the Qubit fluorometer using the Qubit dsDNA HS assay kit. Prepare the sample and perform the measurement according to the manufacturer’s protocol.

3. Use 1 μl of the diluted NET-seq library for characterization on the Agilent Bioanalyzer; use the high-sensitivity DNA analysis kit according to the manufacturer’s instructions.

4. Sequence the human NET-seq library from the 3′ end on the Illumina platform using oLSC006 (Table 2) as a custom sequencing primer.

Transfer the nuclei suspension to a new 1.5 ml RNase-free microcentrifuge tube.

Layer 200 µl of NLB onto nuclei suspension and mix by pulsed vortexing.

Incubate the mixture on ice for 2 min.

Centrifuge at 18,500g for 2 min at 4 °C to collect the chromatin pellet.

Pipette off supernatant from the chromatin pellet. It is important to completely remove the supernatant in order to remove nucleoplasmic RNAs (retain supernatant for the nucleoplasm fraction of western blot, described in Box 1).

Add 50 µl CRS to the chromatin pellet, transfer pellet in CRS to a new 1.5 ml tube and then resuspend pellet in CRS (see Note 28). Perform this step on the experimental sample (for the preparation of nascent RNA, section 3.3.3) and the control sample (for the western blot analysis, as described in Box 1).

Add 700 µl of QIAzol (lysis reagent in miRNeasy mini kit, Qiagen) to the resuspended chromatin (see Note 10 re QIAzol).

Solubilize chromatin pellet by slowly pipetting up and down using a 1-ml syringe with a 22G needle (see Note 29). Mix very carefully (to avoid spilling the sample) until the solution is homogeneous.

Prepare RNA using the miRNeasy mini kit (or miRNeasy micro kit for small cell numbers, see section 2.3.3 step 2) according to the manufacturer’s instructions, including optional DNase treatment.

Measure the prepared RNA quantity and quality using a NanoDrop (see Note 30). The RNA yield is usually in the range of 20–30 µg (this can vary quite a lot depending on the type of cells used). If fractionation was performed on less than 1×107 cells (or RNA yield is less than 20 µg) see Note 24 re RNA amount needed to construct sequencing library. Store the isolated RNA at −80 °C (can be stored for months) until ready to construct sequencing library.

Spray down all work surfaces, pipettes, etc. with RNase away

Denature the RNA sample (from the cellular fractionation, section 3.3.3) and 5 µl of the oGAB11 control (10 µM; Table 1) for 2 min at 80 °C in a Thermomixer. Place on ice.

Prepare linker ligation mix for each RNA sample and for oGAB11 in 0.2-ml RNase-free PCR tubes. Mix until reaction is homogeneous, poor mixing will negatively affect the ligation efficiency.

Add 1 µl Truncated T4 RNA ligase 2 (200U) to each RNA sample as well as the oGAB11 control.

Incubate the ligation mixes for 10 - 16 hours at 16 °C in a thermal cycler (Note 31).

Place on ice and add 0.7 µl of EDTA (0.5 M) to each ligation mix to stop the ligation reaction.

Perform fragmentation calibration test (see Box 2)

Add 20 µl of 2× alkaline fragmentation solution to each sample (from the ligation reaction) and mix. Do not perform fragmentation steps for the oGAB11 ligation control.

Incubate the sample at 95 °C in a thermal cycler for the calibrated time (see Box 2) to fragment RNA (see Note 32).

To purify the fragmented RNA, use the RNA Clean & Concentrator-5 kit (Zymo) and elute RNA from column with 10 uL of 10 mM Tris-HCl (pH 7.0). Alternatively, RNA can be prepared by RNA precipitation (see Note 33).

Prepare RNA ladder and un-ligated oGAB11 control by adding 1.0 µl of RNA control ladder or oGAB11 to 9 µl of RNase-free H2O. Add 10 µl of 2× TBU denaturing sample to the RNA ladder and oGAB11 control, mix well (see Note 34).

Prepare all RNA samples (including the ligated oGAB11 control) by adding 10 µl of 2× TBU denaturing sample buffer to each sample, mix well.

Denature the RNA sample, RNA control samples (ligated oGAB11 and un-ligated oGAB11 controls), and RNA ladder for 2 min at 80 °C. Place the samples on ice.

Pre-run a 15% (wt/vol) polyacrylamide TBE-urea gel at 200 V for 15 min in 1× TBE. Thoroughly flush out gel wells prior to pre-running the gel (see Note 35).

Load gel with the RNA ladder, RNA control samples (ligated oGAB11 and un-ligated oGAB11), and the fragmented RNA sample. Separate by PAGE at 200 V for 65 min.

Stain the gel in 50 ml of gel staining solution for 5 min at room temperature on a shaker (use a black gel box to protect gel from light while staining).

Visualize the gel under blue/UV light.

For the fragmented RNA sample, excise region between 40 and 110 nt. For the ligated oGAB11, excise a narrow band ~55 nt (see Note 36).

Perform rapid gel extraction on the excised gel (see Box 3).

After rapid gel extraction is performed, use the RNA Clean & Concentrator-5 kit (Zymo) and elute RNA from column with 10 uL of 10 mM Tris-HCl (pH 7.0) (at this point, RNA can be stored up to 3 months at -80 °C). Alternatively, RNA can be prepared by RNA precipitation (see Note 33).

Prepare circularization mix and store it on ice.

In a 0.2-ml RNase-free PCR tube, add 4 µl of circularization mix to 15 µl of the cDNA sample and the oGAB11 cDNA control sample, mix well.

Add 1 µl of CircLigase (100 U/µl) and mix.

Incubate the CircLigase reaction for 60 min at 60 °C and then 10 min at 80 °C in a thermal cycler. Circularized cDNA can be stored indefinitely at −20 °C.

Optional: at this point a specific depletion of highly abundant mature RNAs can be performed (see Note

Perform PCR amplification test (Box 4)

Prepare PCR mix for four PCR amplification reactions for both the cDNA sample and the oGAB11 control cDNA sample. Mix well and store on ice.

For each PCR, put 19 µl of PCR master mix in a 0.2-ml RNase-free PCR tube.

Add 1 µl of circularized cDNA and mix it well.

Perform PCR amplifications (as shown in Table 2) with determined optimal number of PCR cycles (see Box 4).

Prepare DNA control ladder. Add 1.0 µl of DNA control ladder to 9 µl of DNase-free H2O. Add 2 µl of 6× DNA loading dye and mix well.

Add 3.4 µl of 6× DNA loading dye to each PCR sample tube and mix well.

Load gel with the PCR products and DNA control ladder. Separate by TBE gel electrophoresis on an 8% (wt/vol) TBE gel at 180 V for 55 min.

Stain the gel in 50 ml of gel staining solution for 5 min at room temperature on a shaker (use a black gel box to protect gel from light while staining).

Visualize the gel under blue/UV light.

For each sample, excise the band from the gel that contains the PCR product. The NET-seq library is a broad band that runs at ~150 nt (use oGAB as baseline and cut up ~50 nt). Avoid contamination from the lower band that runs at ~120 nt, representing PCR product from empty circles (see Note 39). The oGAB11 control sample is not subjected to this step, or any further steps.

Perform the first four steps of rapid gel extraction (see Box 3)

After centrifugation, add 670 µl of DNA soaking buffer and mix.

Incubate the samples overnight at room temperature in a Thermomixer (set at 1,500 r.p.m.).

Use a cut 1,000 µl pipette tip to transfer the gel slurry into a microcentrifuge tube filter.

Centrifuge at 20,000g for 3 min at room temperature. Transfer the eluate to a new 1.5 ml microcentrifuge tube.

Precipitate the NET-seq library by adding 2 µl of 15 mg/ml GlycoBlue and 680 µl of isopropanol. Mix well then incubate the precipitations at −20 °C for ≥1 h (the DNA precipitations can be stored at −20 °C overnight).

Centrifuge at 20,000g for 30 min at 4 °C to pellet the RNA.

Remove the supernatant and wash the pellet with 750 µl of 80% (vol/vol) ice-cold ethanol.

Spin the sample at 20,000g for 2 min at 4 °C, and then remove the supernatant. Air-dry the RNA pellet for 10 min at room temperature.

Resuspend the NET-seq library in 7 - 10 µl of Tris-HCl (10 mM, pH 8.0). The DNA sequencing library can be stored indefinitely at −20 °C.

Prepare a 1:5 dilution of the NET-seq library by adding 1 µl of the NET-seq library to 4 µl of Tris-HCl (10 mM, pH 8.0); mix well (see Note 40).

Use 1 µl of the diluted NET-seq library for quantification with the Qubit fluorometer using the Qubit dsDNA HS assay kit. Prepare the sample and perform the measurement according to the manufacturer’s protocol (see Note 41).

Use 1 µl of the diluted NET-seq library for characterization on the Agilent Bioanalyzer; use the high-sensitivity DNA analysis kit according to the manufacturer’s instructions.

Sequence using oLSC006. Sequence the human NET-seq library from the 3′ end (single-end sequencing) on the Illumina platform using oLSC006 (Table 1) as a custom sequencing primer. Perform sequencing according to the manufacturer’s instructions. NET-seq libraries are typically sequenced on MiSeq, HiSeq or NextSeq next-generation sequencing platforms. A reasonable coverage is obtained with 100–200 million reads.

Below we describe in brief the custom bioinformatics pipeline (see Figure 5) that is used to generate NET-seq coverage files. The scripts are available at http://github.com/churchmanlab/MiMB2019NETseq . See Note 42 for a description of the improvements relative to our previous pipeline (Mayer et al. 2015).

Assess read quality control with FastQC.

Displace 5’ end random barcode sequence from sequence to read identifier line and save as a separate fastq file. This barcode serves as a unique molecular identifier (UMI) and originates from the linker (see Table 1).

Align fastq files with and without UMI sequences separately to a human reference genome using STAR (See Note 43). In case a Drosophila spike-in was used, concatenate the human and Drosophila fasta files to generate a combined reference genome.

Remove alignments resulting from RT mispriming events, i.e. cases where RT priming did not occur at the complementary sequence site on the ligated linker but instead at a position within the RNA fragment. The resulting reads can be identified as they lack the random UMI sequence encoded on the linker and therefore align well to the reference genome both with and without UMI displacement: when the STAR alignment score of a read with the UMI sequence equals the UMI length (10nt) plus the alignment score from that read without UMI.

Remove alignments that map to multiple genomic locations by filtering for their MAPQ value (< 50) using samtools.

Remove alignments resulting from PCR duplication events, i.e. cases of multiple amplicons that originate from the same RNA fragment. The resulting multiple reads are bioinformatically indistinguishable as their alignments share the same position, strand, chromosome, UMI sequence and CIGAR score. To filter out these PCR duplicates, we maintain only one of the alignments.

Remove alignments that could arise from splicing intermediates, i.e. 3’ ends of annotated exons and introns.

Use HTseq to generate a coverage (bedgraph) file containing the read counts at a base-pair resolution over the reference genome as determined from the 5’ end of the read alignments (without UMI) on the opposite strand. The resulting positions correspond to the 3’ ends of nascent RNA and thereby map RNA polymerase positions. The coverage files will be used for a variety of downstream bioinformatics analyses such as visual inspection (e.g. using IGV), metagene profiling (e.g. Bedtools), differential expression (e.g. DEseq2).

The western blot control is used to determine whether or not the cellular fractionation protocol has been executed successfully. The efficiency of the fractionation can be quantified by measuring the amount of elongating Pol II captured in the chromatin fraction (ideally≥95%). When the fractionation protocol is being performed for the first time or being tested on a new cell type, the cytoplasm, nucleoplasm, and chromatin fractions should be checked for subcellular marker proteins (see Note 5) to confirm the fractionation is working (see Note 20).

Adjust the nucleoplasmic (from 3.3.2) and chromatin fraction (from 3.3.2) volumes to the cytoplasmic fraction (from 3.3.1) volume by adding 1× PBS. Mix each fraction well and ensure the chromatin pellet is completely resuspended (see Note 7).

Add 50 µl of 2× SDS buffer (see section 2.3.2 step 7) to 50 µl of each subcellular fraction and boil the samples at 95 °C for 5 min (at this point, samples can be stored for months at −20 °C).

Separate samples by standard SDS-PAGE (loading 10 µl of each boiled sample per lane is usually sufficient).

Probe the membrane overnight at 4 °C with Pol II CTD Ser2-P antibody (3E10, 1:1,000 dilution) and Pol II CTD Ser5-P antibody (3E8, 1:1,000 dilution). These antibodies target transcribing RNA Pol II (see Note 6).

Performing RNA fragmentation results in a homogeneous RNA pool which helps to avoid length biases in any downstream enzymatic reactions. The fragmentation test can be performed using total RNA (or any easily-obtained RNA sample). Once the appropriate fragmentation time is determined (where most of the RNA molecules range from 40 –110 nt), fragmentation can be performed on the nascent RNA (obtained from cellular fractionation) using the determined time (Figure 2); the size range of the nascent RNA and total RNA pools do not vary enough for the fragmentation time to be different.

Take 9 samples of RNA from a total RNA sample (or any other easily obtained RNA sample), each sample should be 20 µl in volume and contain ~1ug of RNA. Add 20 µl of 2× alkaline fragmentation solution to each sample and mix well (see Note 32); fragmentation is not performed for the oGAB11 control.

Fragment each RNA sample at 95 °C in a thermal cycler for the following times: 0, 15, 20, 25, 30, 35, 40, 45, and 50 minutes.

To purify the fragmented RNA, use the RNA Clean & Concentrator-5 kit (Zymo) and elute RNA from column with 10 uL of 10 mM Tris-HCl (pH 7.0). Alternatively, RNA can be prepared by RNA precipitation (see Note 33).

Prepare RNA ladder (denoted as L in figure) by adding 1.0 µl of RNA control ladder to 9 µl of RNase-free H2O. Add 10 µl of 2× TBU denaturing sample to the RNA ladder and mix well.

Prepare all RNA samples by adding 1.0 µl of 2× TBU denaturing sample buffer to each sample, mix well.

Denature the RNA sample and RNA ladder for 2 min at 80 °C. Place the samples on ice.

Pre-run a 15% (wt/vol) polyacrylamide TBE-urea gel at 200 V for 15 min in 1× TBE. Thoroughly flush out gel wells prior to pre-running the gel (see Note 35).

Load gel with the RNA ladder and the fragmented RNA sample. Separate by PAGE at 200 V for 65 min.

Stain the gel in 50 ml of gel staining solution for 5 min at room temperature on a shaker (use a black gel box to protect gel from light while staining).

Visualize the gel under blue/UV light. The optimal fragmentation time is when most RNA molecules are in the required size range, between 40 and 110 nt (the optimal fragmentation time in the figure below is between 25 and 30 minutes).

This protocol for rapidly extracting RNA or cDNA from a polyacrylamide TBE-urea gel is similar to the gel extraction protocol described by the Weissman laboratory (Ingolia et al. 2012; Churchman and Weissman 2012).

Use a 21G needle to pierce the bottom of a 0.5-ml RNase-free microcentrifuge tube.

Place the pierced 0.5-ml tube into a 1.5-ml RNase-free microcentrifuge tube.

Put the gel slice (from fragmentation, RT, or PCR) into the pierced 0.5-ml tube.

Centrifuge the nestled tubes, containing the gel slice, at 20,000g for 4 min at room temperature.

Add 200 µl of RNase-free H2O to the chopped-up gel and mix.

Incubate the sample mixture for 10 min at 70 °C in a Thermomixer.

Vortex the mixture for 30 s at medium intensity.

Cut the tip off of a 1,000-µl pipette tip and transfer the gel mixture into a microcentrifuge tube filter.

Centrifuge the mixture at 20,000g for 3 min at room temperature.

The NET-seq library is PCR amplified using an Illumina index forward primer (Barcode primer, see Table 1) and a reverse primer (oNTI231; Table 1) specific to NET-seq (Figure 4). For each NET-seq library preparation, the minimum number of PCR cycles needed to obtain a sequencing library must to be determined. The NET-seq library is then amplified with the determined minimal amount of PCR cycles; this avoids overamplification which can result in unwanted products such as PCR duplicates.

Prepare the PCR master mix (see section 2.4.8 step 4) for four pilot PCR amplifications for each cDNA sample and the oGAB11 cDNA control sample (denoted as O in figure). Mix well and store it on ice.

For each PCR sample, put 19 µl of PCR master mix in a 0.2-ml RNase-free PCR tube.

Add 1 µl of circularized cDNA and mix it well.

Perform PCR pilot amplifications (described in Table 2). Remove one PCR tube for each sample at the end of the extension step after 6, 8, 10 and 12 amplification cycles.

Add 3.4 µl of 6× DNA loading dye to each tube and mix well.

Prepare DNA control ladder (denoted as L in figure) by adding 1.0 µl of ladder to 9 µl of DNase-free H2O. Then add 2 µl of 6× DNA loading dye and mix well.

Separate the PCR products and the DNA control ladder by TBE gel electrophoresis on an 8% (wt/vol) TBE gel at 180 V for 55 min.

Stain the gel in 50 ml of gel staining solution (see section 2.4.8) for 5 min at room temperature on a shaker (use a black gel box to protect gel from light while staining).

Visualize the gel under blue/UV light and identify the optimal PCR amplification cycles for each cDNA sequencing library. The optimal PCR amplification cycles for the NET-seq library, which runs at ~150 nt, is determined by the presence of a clear band at ~150 nt and no higher-molecular-weight PCR products (the optimal number of cycles in the figure below is between 6 and 8).