title: RNA-seq of DNA-templated transcription reaction
design: This customized RNA-seq protocol is derived from the TruSeq Small RNA Library Preparation Kit (Illumina) and AQ-seq (PMID 30605524). Key changes from the commercial TruSeq kit include 1) buffer composition of the ligation reaction; 2) randomization of an adaptor sequence to add unique molecular identifiers; and 3) reverse transcription and PCR enzymes. These changes reduce per-reaction cost by ~10-fold and ligation bias without substantially impacting hands-on time. All commercial kits were used as directed unless otherwise specified. The 3' adaptor (/5Phos/TGGAATTCTCGGGTGCCAAGG/3ddC/) was enzymatically adenylated with the 5' DNA adenylation kit (New England Biolabs; NEB). The adenylation reaction was purified with the Oligo Clean & Concentrator-5 kit (Zymo Research; with two wash steps, instead of one, and with an extra spin to dry the column before elution), eluted in 15 L IDTE (pH 8.0; Integrated DNA Technologies; IDT), and diluted to 6 M. The 3' adaptor ligation reaction contained 0.6 M adenylated adaptor (3 pmol), 20,000 U/mL T4 RNA Ligase 2, truncated K227Q (NEB; 100 U), 20% PEG-8000, and 1x T4 RNA Ligase Buffer (NEB) in a total volume of 5 L. 1.5 L RNA (eluate from the purification of RNAP extension reactions (~2 M RNA oligonucleotide template) or the DNA-templated transcription reaction) was combined with the adaptor in a total volume of 2 L. The RNA-adaptor mix was heated at 72C for 2 min and cooled on ice. Then, 3 L of solution containing enzyme, PEG-8000, and buffer was added. The reaction was incubated at 25C for 1 hr. Then, STP oligonucleotide (GAAUUCCACCACGUUCCCGUGG) was added to a final concentration of 1.8 M in a volume of 0.5 L (10 pmol), and the reaction was incubated for a further 15 min at 25C. The 5' adaptor ligation reaction contained 1.4 M 5' adaptor (GUUCAGAGUUCUACAGUCCGACGAUCNNNN; 10 pmol), 714 U/mL T4 RNA ligase 1 (NEB; 5 U), 1 mM ATP, 16.3% PEG-8000, and 1x T4 RNA Ligase Buffer (NEB) in a total volume of 7 L. The 5' adaptor was heated at 72C for 2 min and cooled on ice. Then, 1.5 L of solution containing adaptor, enzyme, ATP, PEG-8000, and buffer was added to the 5.5 L 3' ligation reaction:STP oligonucleotide mixture; the resulting 5' ligation mixture was incubated for 1 hr at 25C. The reverse transcription (RT) reaction contained 14,814 U/mL Protoscript II (NEB; 200 U), 460 M dNTPs, 7.41 mM DTT, and 0.74x Protoscript II buffer. To the 7 L of unpurified ligation reaction, RT primer (GCCTTGGCACCCGAGAATTCCA) was added to 0.5 M in a volume of 1 L (4 pmol). The RNA:primer mixture was heated at 72C for 2 min and cooled on ice. Then, 5.5 L of solution containing enzyme, dNTPs, DTT, and buffer was added. The reaction was incubated at 42C for 1 hr. The PCR reaction contained 0.24 M universal primer (AATGATACGGCGACCACCGAGATCTACACGTTCAGAGTTCTACAGTCCGA; 12 pmol), 0.8 M indexing primer (see TruSeq small RNA kit for sequences; 40 pmol), 1x NEBNext High-Fidelity 2X PCR Master Mix (NEB), and the entire volume of the RT reaction in a total volume of 50 L. For some experiments, we used indexing primers from the TruSeq Small RNA Library Preparation Kit (2 L of primer). The source of the indexing primer did not substantially impact sequencing results. 25 L of PCR reaction was amplified with 12 cycles of PCR (98C for 10 sec, 60C for 30 sec, and 72C for 30 sec; initial denaturation at 98C for 30 sec; final extension at 72C for 15 min). The libraries were loaded on a 2% agarose gel and run for ~2 hr at 5 V/cm. DNA in the range ~125-300 nt (corresponding to RNA fragments ~0-175 nt) was excised with a razor blade. For the library prepared from the DNA-templated transcription reaction, DNA in the range ~400-500 nt was excised. Libraries were purified with the Zymoclean Gel DNA Recovery Kit (Zymo Research; with an extra spin to dry the column before elution); elution was in 6 L IDTE (pH 8.0). Libraries were quantified via the Qubit dsDNA High Sensitivity Assay (ThermoFisher Scientific).