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 μL IDTE (pH 8.0; Integrated DNA Technologies; IDT), and diluted to 6 μM. The 3' adaptor ligation reaction contained 0.6 μM 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 μL. 1.5 μL RNA (eluate from the purification of RNAP extension reactions (~2 μM RNA oligonucleotide template) or the DNA-templated transcription reaction) was combined with the adaptor in a total volume of 2 μL. The RNA-adaptor mix was heated at 72°C for 2 min and cooled on ice. Then, 3 μL of solution containing enzyme, PEG-8000, and buffer was added. The reaction was incubated at 25°C for 1 hr. Then, STP oligonucleotide (GAAUUCCACCACGUUCCCGUGG) was added to a final concentration of 1.8 μM in a volume of 0.5 μL (10 pmol), and the reaction was incubated for a further 15 min at 25°C. The 5' adaptor ligation reaction contained 1.4 μM 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 μL. The 5' adaptor was heated at 72°C for 2 min and cooled on ice. Then, 1.5 μL of solution containing adaptor, enzyme, ATP, PEG-8000, and buffer was added to the 5.5 μL 3' ligation reaction:STP oligonucleotide mixture; the resulting 5' ligation mixture was incubated for 1 hr at 25°C. The reverse transcription (RT) reaction contained 14,814 U/mL Protoscript II (NEB; 200 U), 460 μM dNTPs, 7.41 mM DTT, and 0.74x Protoscript II buffer. To the 7 μL of unpurified ligation reaction, RT primer (GCCTTGGCACCCGAGAATTCCA) was added to 0.5 μM in a volume of 1 μL (4 pmol). The RNA:primer mixture was heated at 72°C for 2 min and cooled on ice. Then, 5.5 μL of solution containing enzyme, dNTPs, DTT, and buffer was added. The reaction was incubated at 42°C for 1 hr. The PCR reaction contained 0.24 μM universal primer (AATGATACGGCGACCACCGAGATCTACACGTTCAGAGTTCTACAGTCCGA; 12 pmol), 0.8 μM 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 μL. For some experiments, we used indexing primers from the TruSeq Small RNA Library Preparation Kit (2 μL of primer). The source of the indexing primer did not substantially impact sequencing results. 25 μL of PCR reaction was amplified with 12 cycles of PCR (98°C for 10 sec, 60°C for 30 sec, and 72°C for 30 sec; initial denaturation at 98°C for 30 sec; final extension at 72°C 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 μL IDTE (pH 8.0). Libraries were quantified via the Qubit dsDNA High Sensitivity Assay (ThermoFisher Scientific).