Name: GSM8624752
Instrument: Illumina NovaSeq X Plus
Strategy: OTHER
Source: OTHER
Selection: other
Layout: SINGLE
Construction protocol: To harvest nascent transcripts for the NET-seq workflow, cultures were filtered between two vacuum filtration systems using a 0.45 µm pore nitrocellulose filter (GVS Micron Sep, 1215305). Cells were scraped off each filter using a spatula and plunged immediately into liquid nitrogen (i.e., cells from the same culture were combined into the same 50 mL conical tube containing ~25 mL liquid nitrogen). Collected cells were cryo-lysed using a RETSCH mixer mill (MM 400) as previously described (Larson, M. H. et al. 2014. Science 344: 1042-47), with the exception that 50 mL stainless steel canisters and a 25 mm stainless steel ball were used to perform the cryomilling. To isolate nascent transcripts, we performed a modified 3xFLAG-IP protocol with previously described buffers Larson, M. H. et al. 2014. Science 344: 1042-47). Specifically, the thawed grindate volume was scaled to 5.5 mL with lysis buffer (1x lysis stock [20 mM Tris, pH 8.0, 0.4% Triton X-100, and 0.1% NP-40 substitute], 100 mM NH4Cl, 1x EDTA-free cOmplete Mini protease inhibitor cocktail [Roche Diagnostics GmbH, 11836170001], 10 mM MnCl2, and 50 U/mL RNasin [Promega, N211B], and 0.4 mg/mL puromycin), DNA was partially digested for 20 min with RQ1 DNase (0.054 U/mL [0.02 U/mL for the E. coli-only NET-seq pilot experiment])[Promega, M6101], and digestion reactions were stopped by addition of EDTA to 28 mM (final concentration). RNAP-nascent transcript complexes were directly immunoprecipitated using Anti-FLAG M2 affinity gel (Sigma, A2220) (i.e., without buffer exchange), and the precipitated RNAP-nascent transcript complexes were subsequently washed four times (1x lysis stock, 100 mM NH4Cl, 300 mM KCl, 1 mM EDTA, and 50 U/mL RNasin) [Promega, N2515]. RNAP-nascent transcript complexes were eluted twice with 3xFLAG peptide (Sigma, F4799) (1x lysis stock, 100 mM NH4Cl, 2 mg/mL 3xFLAG peptide, 1 mM EDTA, and 50 U/mL RNasin). Nascent transcripts were purified using a miRNeasy kit [Qiagen, 217084] as previously described (Larson, M. H. et al. 2014. Science 344: 1042-47). However, to reduce phenol and chaotropic salt contamination, nascent transcripts were subjected to an additional overnight isopropanol-GlycoBlue (Invitrogen, AM9516) precipitation at -20 °C. For nascent transcript library generation, we followed a modification of a previous NET-seq workflow (Larson, M. H. et al. 2014. Science 344: 1042-47 and Churchman, L. S. & Weissman, J. S. 2011. Nature 469: 368-73). Specifically, our workflow included using custom adaptors compatible with an Illumina NovaSeq X instrument. Likewise, the DNA adapter used for nascent transcript 3' end ligation was adenylated using components from a NEB 5' DNA Adenylation kit (E2610; 6µM DNA linker [/5Phos/NNNNNNNNNNgcagctCTGTAGGCACCATCAATGATCGTCGGA/3ddC/], 80 µM ATP, 6 µM Mth RNA ligase, and 1X Adenylation Reaction Buffer). The adenylation reaction was incubated for 4 h incubation at 65 °C, inactivated at 85 °C for 5 min, and precipitated overnight at -20 °C with isopropanol and GlycoBlue (Invitrogen AM9516). The precipitated, adenylated DNA linker was ligated to 750 ng of precipitated nascent transcripts, in duplicate, using components of a NEB T4 RNA Ligase 2, truncated (T4 Rnl2tr) kit (M0242; 10% DMSO, 22% PEG8000, 3 µM adenylated DNA linker, T4 Rnl2tr [14.7 U/µL], RNasin [2U/µL], and 1x T4 RNA Ligase Reaction Buffer). These ligation reactions were incubated at 37 °C for 4 h. After this incubation, T4 Rnl2tr was inactivated by incubation with Proteinase K (0.04 U/µL) (NEB, P8107) at 37 °C for 1 h. RNAs were fragmented, resolved, gel extracted, and precipitated as previously described (Larson, M. H. et al. 2014. Science 344: 1042-47 and Churchman, L. S. & Weissman, J. S. 2011. Nature 469: 368-73), with the exception that the gel extraction incubation at 70 °C was increased to 25 min. cDNAs were synthesized using a custom adapter (/5Phos/AGATCGGAAGAGCACACGTCTGAAC/iSp18/CACTCA/iSp18/CCTACACGACGCTCTTCCGATCTTCCGACGATCATTGATGGTGCCTACAG) and a previously described protocol (Larson, M. H. et al. 2014. Science 344: 1042-47 and Churchman, L. S. & Weissman, J. S. 2011. Nature 469: 368-73), with the exception that the reaction time was increased to 1 h. Circularization of gel extracted and precipitated cDNAs was performed using a protocol previously described (Larson, M. H. et al. 2014. Science 344: 1042-47 and Churchman, L. S. & Weissman, J. S. 2011. Nature 469: 368-73), with the exception that the circularization reaction incubation period was increased to 3 h and the gel extraction incubation period was increased as above. After circularization, cDNA libraries were PCR amplified using minimal cycles and custom adapters (AATGATACGGCGACCACCGAGATCTACACAGCGAGCTACACTCTTTCCCTACACGACGCTCTTCCGATCTTCCGACGATC and CAAGCAGAAGACGGCATACGAGATAAGGATGAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT), gel extracted, and precipitated as previously described (Larson, M. H. et al. 2014. Science 344: 1042-47 and Churchman, L. S. & Weissman, J. S. 2011. Nature 469: 368-73). Library concentration and amplified product size distribution were determined using an Agilent TapeStation 4150. NET-seq libraries were sequenced by the University of Wisconsin-Madison Biotechnology Center on an Illumina NovaSeq X Plus instrument.