2008, Gregersen et al., 2014, and Heyn et al., 2014, with modifications, such as extension to

Transcription

1 DETAILED PROTOCOL s 4 U-RNA enrichment with MTS chemistry This protocol uses MTS chemistry and builds upon previous methods developed by Dölken et al. 2008, Gregersen et al., 2014, and Heyn et al., 2014, with modifications, such as extension to mirna analysis, as noted in the protocol. 1. Biotinylation: In a microfuge tube, combine RNA (50 µg total) with biotinylation buffer (final conditions: 10 mm HEPES [ph 7.5], 1 mm EDTA) and MTSEA biotin-xx (5 µg Biotium) dissolved in DMF (final concentration of DMF = 20%) in a total of 150 µl. Wrap tubes in aluminum foil and incubate at room temperature for 2 hr with rotation. 2. Cleanup: Excess MTS-biotin can be removed by phenol:chloroform extraction followed by isopropanol precipitation. Alternatives include RNeasy Mineulte Kit (Qiagen) for RNA >200 nt (elute into 50 µl instead of the recommended 14 µl). For RNAs <200 nt, yields are improved by using a Nucleotide Cleanup Kit (Qiagen) or mirvana kit (Life Technologies) following the manufacturer s instructions. At this step, purified RNA should be brought up to 50 µl in water regardless of purification method. 3. Separation on streptavidin beads: Comparable yields were found when biotinylated s 4 U-RNA was separated from non-biotinylated RNA using µmacs Streptavidin Microbeads (Miltenyi) and Dynabeads Streptavidin C1 beads (Invitrogen). Both protocols are included below. µmacs Streptavidin Microbeads (Miltenyi): a. To 50 µl biotinylated s 4 U-RNA, add 200 µl bead solution and incubate for 15 min at room temperature in the dark with rotation.

2 b. In the meantime, place one µcolumn per reaction in the magnetic field of the µmacs separator and equilibrate with 2 x 200 µl nucleic acid equilibration buffer supplied with the beads. c. Apply the reactions to the µcolumns and collect the flow-through in a fresh microfuge tube. d. Wash µcolumns twice with 500 µl high salt wash buffer (100 mm Tris-HCl [ph 7.4], 10 mm EDTA, 1 M NaCl, and 0.1% Tween-20). e. Elute s 4 U-RNA from µcolumns with 100 µl freshly prepared 100 mm DTT, collect the eluted RNA, wait 5 min, and elute with an additional 100 µl DTT solution, combining the eluted s 4 U-RNA (200 µl total). Dynabeads Streptavidin C1 beads (Invitrogen): a. Aliquot 100 µl beads (10 mg/ml) per reaction into microfuge tube and place on a magnetic rack for 2 min. Discard the supernatant. b. Wash beads with 2 x 500 µl RNase-free water, followed by separation on a magnetic rack as in step a. c. Apply 100 µl biotinylated s 4 U-RNA to beads along with 10 µl high salt wash buffer and incubate at room temperature in the dark with rotation for 1 hr. d. Separate the beads on a magnetic rack, transfer the supernatant into a fresh microfuge tube and save this sample as flow-through. e. Wash beads twice with 500 µl high salt wash buffer (100 mm Tris-HCl [ph 7.4], 10 mm EDTA, 1 M NaCl, and 0.1% Tween-20). f. Elute the s 4 U-RNA from beads by resuspending the beads in 100 µl freshly prepared 5% β- mercaptoethanol (β-me) at room temperature, incubate 15

3 min at room temperature in the dark. Separate the beads, and collect the supernatant. Resuspend beads in 100 µl β-me at 50 C and collect the supernatant immediately. Combine both supernatants and save this sample as the enriched s 4 U-RNA. 4. RNA cleanup: Recover RNA from the flow-through and eluent fractions using the MinElute Spin columns (Qiagen) according to the instructions of the manufacturer, or for RNAs <200-nt, using ethanol precipitation. Samples are now ready for analysis via urea- PAGE or for library preparation prior to deep sequencing. Modeling expected yields The objective of the experiment described above is to determine the fraction of newly transcribed RNA (f i ) for each RNA in the cell. The output of the experiment is the yield of each RNA over input (X i ): where X i can be related to f i : X! = RNA!"#$%!!" RNA!"#$% X! = f! p! (N! ) Longer RNAs generally have a higher number of uridines and are therefore more likely to be captured. Specifically, the probability of capture, p i (N i ), is a function of the number of uridine residues (N i ) in the transcript. In order for X i f i, we need p i (N i ) to be as close to unity as possible. We can model the length dependence of p i (N i ) as follows: p! N! =!!! 1 1 y!"# p(u! = j)!!! where y bio is the yield of s 4 U biotinylation, and U i is the number of s 4 U residues in the transcript.

4 If we assume that s 4 U residues are randomly incorporated into newly transcribed RNA at sites of uridine, then U i will display a binomial distribution with mean r, the ratio of s 4 U to uridine r =!!!!!"! incorporated into the RNA according to the following equation: p U! = j = N! j r! (1 r)!!!! This model assumes that any RNA with one or more biotins will be retrieved quantitatively, which agrees well with the high affinity of streptavidin for biotin, and the observation that the flow through does not contain significant amounts of biotinylated RNA (see Figure S1F). We have modeled the expected yields of biotinylation at given level of s 4 U incorporation (r = 0.075) and the results agree well with the experimental data from enrichment using an in vitro transcribed RNA ladder (Figure S1G), supporting the utility of this model. Based on this model, there are two ways to decrease the length bias: (1) increase y bio, the yield of conversion of s 4 U to bio-s 4 U, or (2) increase r, the number of s 4 U s in the transcript. This manuscript describes MTS-chemistry that dramatically increases y bio, the yield of biotinylation of each s 4 U residue. In agreement with our data and these equations, this improvement leads to higher yields of s 4 U-RNA (Figure 2B, C) and lower length bias (Figure 2D and Figure S1F). For (2), increasing the [s 4 U] fed to cells can increase r. The extent to which r can be increased has practical constraints. At very high [s 4 U], the nucleoside is toxic to the cells (Burger et al., 2013). In one case (Heyn et al., 2014), it was possible to further increase incorporation by directly injecting s 4 UTP into cells, which provided high enrichment of even short transcripts. However, direct injection of individual cells is not always feasible. We find that under the highest commonly used concentrations of nucleoside ([s 4 U] = 700 µm), even the longest spliced transcripts are enriched at lower levels with HPDP-biotin than with MTS-biotin (Figure 2D). In other words, under standard incorporation rates, the difference in y bio between

5 MTS-biotin and HPDP-biotin has a significant impact on X i. Irrespective of the incorporation rate, it is always preferable to increase the yield of biotinylation (y bio ) to make more efficient use of the s 4 U that has been incorporated into the labeled RNA. It is interesting to note that according to this model, low yields of biotinylation (such as those achieved using HPDP-biotin) lead to comparative enrichment of very long transcripts (such as those containing long, unspliced introns) over moderately sized transcripts. While low sequencing coverage in the input samples prevented accurate quantitation of X i for these long, low-abundance, unspliced transcripts, this enrichment is clearly evident in the mapped coverage, consistent with this prediction. It is also worth noting that measurements of RNA half-lives using HPDP-biotin have the potential to be accurate (provided sufficient signal-to-noise) because the length bias is constant for any given RNA over time (Neymotin et al., 2014). The relative amounts of different transcripts and the analysis of splicing, however, may be strongly influenced by the length bias of capture. The low y bio for HPDP-biotin is expected to influence these results. An example of this effect can be seen in Figure 2E. Troubleshooting tips 1. While we have found a 50-fold molar excess of biotinylation reagent to be successful under the conditions described above, we still recommend titratating the amount of MTSbiotin used until saturation is reached (an example is found in supplementary Figure S1H). 2. A loading control is strongly recommended to control for variability in sample preparation and to be used for downstream normalization between samples in RNA-Seq experiments.

6 Appropriate internal controls for long RNAs (>200-nt) include S. pombe RNA; for small RNAs (<200-nt), synthetic RNAs can be used, such as those in Table S3. 3. To ensure conditions are appropriate for successful enrichment, we recommend preforming positive and negative control experiments in parallel to experimental samples. One convenient control is to use an in vitro transcribed ladder (RNA Century Plus Marker Template and Maxiscript T7 transcription kit, Invitrogen) with s 4 UTP included in the IVT reaction (positive control) or without s 4 UTP (negative control). Including fluorescent nucleotide triphosphates in these reactions allows for convenient and sensitive detection of the enriched RNA after PAGE. For examples of these experiments, see Figure S1F.