PRO-seq assay and analysis

The protocol was adapted from Kwak et al.⁵⁰. Drosophila embryos of 3.5–4.5 h age were dechorionated and homogenized in 1 ml of buffer A (10 mM Tris-Cl pH 8.0, 300 mM sucrose, 3 mM CaCl₂, 2 mM MgAc₂, 0.1% Triton X-100, 0.5 mM DTT). The homogenate was filtered through a nylon membrane (pore size 0.8 µm) and spun down at 500×g. The pellet was resuspended in fresh buffer A and spun down several times for washing. Nuclei were then resuspended in buffer D (10 mM Tris-Cl pH 8.0, 25% glycerol, 5 mM MgAc₂, 0.1 mM EDTA, 5 mM DTT) to achieve a final concentration of roughly 106 cells per 5 µl before freezing in liquid nitrogen. Run-on reaction was performed for 3 min at 30 °C after adding 2x reaction mix to the samples (10 mM Tris-Cl pH 8.0, 5 mM MgCl₂, 1 mM DTT, 300 mM KCl, 0.05 mM of each of the four Biotin-NTP, 0.4 U/µl RNAse inhibitor, 1% sarkosyl). Nascent RNA was extracted directly after with Trizol, precipitated in ethanol, and fragmented by base hydrolysis in 0.2 N NaOH on ice for 10 min. After neutralization with 1 M Tris-Cl pH 6.8, salts and free NTPs were removed by buffer exchange on a P-30 column (Bio-Rad). To enrich biotin-labeled RNA, the samples were bound to 30 µl M280 streptavidin beads (Invitrogen) according to the manufacturer and washed with twice with high salt buffer (2 M NaCl, 50 mM Tris-Cl pH 7.4, 0.5% Triton X-100), twice with binding buffer (300 mM NaCl, 10 mM Tris-Cl pH 7.4, 0.1% Triton X-100) and once with low salt buffer (5 mM Tris-Cl pH 7.4, 0.1% Triton X-100). RNA was extracted from the beads twice with 300 µl Trizol in each round and precipitated with glycoblue in ethanol. RNA pellets were redissolved in 4 µl of 12.5 µM reverse 3′ adaptor dilution (Rev3, see Supplementary Table ⁹) and ligated overnight at 4 °C with T4 RNA ligase (NEB) in 10 µl reaction volume (1x T4 RNA ligase buffer, 1 mM ATP, 10% PEG, 1U/µl RNAse inhibitor). The bead binding and Trizol extraction was repeated as above and 5′-decapping performed using RppH (NEB) by dissolving pellets in 10 µl H₂O, adding 40 µl of the reaction mix (1x NEBuffer 2, 2 U/µl RppH, 2 U/µl RNAse inhibitor) and incubating at 37 °C for 1 h. 5′-hydroxyl was performed by directly adding PNK (NEB) reaction mix to each decapped sample. The total reactions of 100 µl volume (1x PNK buffer, 1 mM ATP, 2.5 µl PNK, 0.2 U/µl RNAse inhibitor) were incubated at 37 °C for 1 h. The RNA was then extracted using Trizol and precipitating in ethanol. Pellets were redissolved in 4 µl of 12.5 µM reverse 5′ adaptor (VRA5, see Supplementary Table ⁹) and ligation performed as with the 3′ adaptor. The RNA was enriched again by bead binding and extraction as above and resuspended in 10 µl H₂O. Reverse transcription was performed using SuperScript III (Invitrogen) in 20 µl of final reaction volume. Water, dNTPs (final 0.5 mM) and reverse transcription primer (RP1 TrS, see Supplementary Table, final 2.5 µM) were added first, samples heated to 70 °C for 2 min, chilled on ice for 2 min and the remaining reaction components except for the enzyme added (1x first-strand buffer, 5 mM DTT, 1 U/µl RNAse inhibitor). After incubation at 37 °C for 5 min, the enzyme was added (15 U/µl) and the reaction run in a PCR machine: 45 °C for 15 min, 50 °C for 40 min, 55 °C for 10 min, 70 °C for 15 min. The ideal amount of amplification cycles was determined by makig serial dilutions of the template and test amplification with primer RP1 TrS, primer RPI TrS short (see Supplementary Table ⁹) and Phusion polymerase 2x master mix (Thermo Fisher). PCR cycling: 95 °C – 2 min, 5 × [95 °C – 30 s, 56 °C – 30 s, 72 °C – 30 s], 9 × [95 °C – 30 s, 65 °C – 30 s, 72 °C – 30 s], 72 °C – 10 min. Each library dilution row was run on a 8% polyacrylamide gel, and the ideal cycle number determined by choosing the amplification that produced a visible but not overamplified 125–350 bp fragment library. Full-scale amplification was then performed using barcoded reverse primers (RPIx, see Supplementary Table ⁹). The amplified libraries were purified using 1.8x volume ratio AMPure XP beads (Agencourt). The libraries were then separated on a 0.5× TBE 8% polyacrylamide gel and cut from around 125 bp (just above visible primer-dimer) to 350 bp. The samples were extracted from the gel fragments by shredding the fragments and incubating them in twice the amount of extraction buffer (10 mM Tris-Cl pH 8, 0.5 mM NaAc, 10 mM MgAc₂, 1 mM EDTA, 0.1% SDS) at 50 °C for 13 h. After spinning down at 10,000xg for 5 min and retrieving the eluted library, a second extraction round was performed with 600 µl buffer for 2 h and the supernatants pooled. Eluates and remaining gel fragments were filtered through Spin-X filters (Corning), and the volume reduced to 500 µl in a speed vacuum centrifuge. The DNA was then extracted using buffered phenol–chloroform, and the concentration measured. Barcoded libraries were pooled and sequenced on the Illumina Hiseq2500 platform with 1 × 50 bp SR configuration.

The adaptors were first trimmed from the sequencing reads by cutadapt software and the reads with at least 15 nt were retained. We then removed reads that mapped to rRNAs, and the remaining reads were further mapped to the Drosophila melanogaster (dm3) genome assembly using BWA with the default parameters. We also generated the PRO-seq coverage tracks (normalized by the library sizes) with separate strands for each factor. To detect de novo transcripts from PRO-seq, we combined all genotypes and adapted the Homer2⁵¹ GRO-seq transcript identification method (using a parameter setting “findPeaks -style groseq -tssFold 4 -bodyFold 3”). The pausing regions (promoter region) were defined from the de novo transcript starts to 200 nt downstream, and the gene-body regions were defined from 400 nt downstream to the end of the de novo transcripts. The de novo transcripts having a promoter expression of greater than 1 transcript per million (TPM) were retained for further analysis. Fold difference between adjacent gene pairs is summarized in Supplementary Data ⁵.