1. Irritant, handle with care and dispose of the waste according to institutional regulations.
2. Toxic, handle with care and dispose of the waste according to institutional regulations.
3. Irritant, harmful, and hazardous to the environment: handle with care and dispose of the waste according to institutional regulations.
4. Toxic, corrosive, and an irritant: handle with care and dispose of waste according to institutional regulations.
5. When the fractionation protocol is being tested on a new cell type it is recommended that the cellular fractions are checked for subcellular marker proteins in order to confirm that the fractionation is working. To do this, probe the WB membrane with antibodies raised against GAPDH (cytoplasm), the U1 snRNP70 (nucleoplasm) and histone 2B (chromatin).
6. The CTD specificities of these Pol II–specific antibodies have been extensively characterized, so we recommend using these specific antibodies when testing the fractionation protocol.
7. Benzonase (250 U) can be used to resuspend the sticky chromatin pellet in solution (for the WB control, Box 1).
8. Flammable, corrosive, and an irritant: handle with care and dispose of waste according to institutional regulations
9. Volatile, toxic, and an irritant: handle with care and dispose of waste according to institutional regulations.
10. It is important to use the miRNeasy mini kit (or miRNeasy micro kit for small cell numbers) instead of the RNeasy mini kit to avoid RNA length biases. Use personal protective equipment when handling reagents in the kits; the RWT buffer is corrosive and an irritant, and the QIAzol lysis reagent contains phenol and is toxic and corrosive. Dispose of waste according to institutional regulations.
11. Handle sharps with care and dispose according to institutional regulations.
12. Keep track of the enzyme lot; ligase activity can vary between different batches.
13. Using an N10 linker increases the dynamic range of the NET-seq library and decreases PCR duplicates (compared to the N6 linker used in (Mayer and Churchman 2016)). 14. Use personal protective equipment when handling reagents in the kit; buffers are irritants. Dispose of waste according to institutional regulations.
15. Nucleic acid stains are usually mutagenic; use personal protective equipment when handling. SYBR Gold stain is flammable; dispose of waste according to institutional regulations.
16. Corrosive and an irritant: handle solutions with care and dispose of waste according to institutional regulations. Limit the freeze-thaw cycles for this reagent.
17. Toxic and hazardous to the environment: handle with care and dispose of waste according to institutional regulations.
18. Highly flammable, volatile, and an irritant: handle solutions with care and dispose of waste according to institutional regulations.
19. NET-seq has been successfully carried out on several human cell lines (e.g. K562, HeLa S3, HEK293T, and MOLT-4 cells), including challenging cell types such as neurons. The NET-seq protocol has also been successfully applied to other cell types such as Drosophila cells (S2 and S2R+ cells) and mouse cells (NIH 3T3 cells) and others.
20. Perform a western blot control in parallel with the sample, or prior to performing fractionation on the sample to ensure the cellular fractionation protocol is optimized for the specific tissue being used. The western blot control is a separate sample from the sample being prepared to make a sequencing library.
21. We used roughly 1/2 of a liver per sample.
22. If there is a small amount of supernatant remaining in the tube after the greater quantity has been removed, it is helpful to use a small pipette tip to get rid of the residual supernatant.
23. For more effective layering, tilt the tube at a 45 degree angle and pipette lysate onto the side of the tube.
24. Some chromatin pellets can be difficult to resuspend. If you find this to be the case, you can either increase the volume of the CRS (up to 300 µl) and try to solubilize the chromatin pellet again or you can proceed directly to the RNA extraction (Steps section 1.3.3) by adding Qiazol to your chromatin pellet (which is in 50 µl of CRS).
25. Alternatively, the chromatin pellet can also be solubilized by gentle vortexing.
26. The expected absorbance at (A260/A280) ratio is ~2.0, indicative of highly purified RNA.
27. The fragmentation time needs to be adjusted whenever a new batch of alkaline fragmentation solution is applied (or when the solution has not been used for long periods of time -i.e. months). 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.
28. The un-ligated oGAB11 control is only used for visualizing the efficiency of the ligation reaction by comparing un-ligated oGAB11 to ligated oGAB11. After visualization of the gel, un-ligated oGAB11 control is not subjected to any further steps in the protocol.
29. Use a needle and syringe to flush out the excess urea that may settle in the gel wells prior to loading samples.
30. The efficiency of the linker ligation is usually >95%, the efficiency of the ligation is monitored using the oGAB11 control by PAGE (see Figure 2).
31. If no cDNA smear is observed continue to the PCR step; sometimes PCR product will be visible even when the RT product is not visible by eye. Other possible reasons for not observing the RT product are that the linker ligation was inefficient or the reverse transcriptase enzyme lost activity. One way to prevent these problems is to replace the ligation reaction and RT reaction reagents every 4 months.
32. Empty circles are circularized DNA molecules that arise from un-extended RT primers, and hence they do not contain any information about the original nascent RNA.
33. Do not dilute the NET-seq library for the quantification and characterization step if less than 1 µg of starting material was used to construct the sequencing library.
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