Biotin 11 ctp

The genomic region chr19:45406985–45408892 (on genome build hg19) was cloned from human fibroblast DNA into the pcDNA3.1-GFP(1–10) (Addgene cat# 70219) vector. The clone sequence was as follows:
The sequence includes 691 distinct 5-mers, about 70% [691/(4⁵)] of the 5-mers of the RNA synthesized with just the canonical nucleotides.
We used the MEGAscript T7 transcription kit (ThermoFisher, cat# AM1334) for in vitro transcription (IVT) in the presence of varying amounts of modified nucleotides to prepare RNA for Nanopore sequencing. The modified nucleotides used were the 2′-O-methyl-nucleotide set (TriLink Biotechnologies, cat# K-1012), N6-methyladenosine-5′-triphosphate (TriLink Biotechnologies, cat# N-1013-1), N1-methyladenosine-5′-triphosphate (TriLink Biotechnologies, cat# N-1042-1), 5-methylcytidine-5′-triphosphate (TriLink Biotechnologies, cat# N-1014-1), 5-hydroxymethylcytidine-5′-triphosphate (TriLink Biotechnologies, cat# N-1087-1), pseudouridine-5′-triphosphate (TriLink Biotechnologies, cat# N-1019-1), and biotin-11-CTP (Perkin-Elmer, cat# NEL542001EA). In vitro-transcribed RNAs were purified from their reaction mixes using RNAClean XP beads (Beckman Coulter, cat# A63987), and the integrity of the RNA (∼2 kb, and no evidence of degradation) was verified using the Agilent RNA 6000 Nano Kit (cat# 5067-1511).

BEAS-2B, A549, and LC-2/ad cells were each plated on 2 × 15 cm tissue culture dishes and grown to confluence. Cells were harvested and nuclei prepared as described (22 (link)). Aliquots containing 10E6 nuclei in 100 μL Freezing Buffer (50 mM Tris-HCl [pH 8.3], 5 mM MgCl₂, 40% glycerol, 0.1 mM EDTA [pH 8.0], 4 U/mL SUPERase-In) were flash frozen in a dry ice/ethanol bath and stored at –80°C. After briefly thawing on ice, 100 μL aliquots of 10E6 nuclei were added to 100 μL of Reaction Buffer (5 mM Tris-HCl [pH 8.0]; 2.5 mM MgCl₂; 0.5 mM DTT; 150 mM KCl; 0.025 mM each of Biotin-11-CTP [PerkinElmer] and ribonucleoside CTP; 0.125 mM each of ribonucleoside ATP, ribonucleoside GTP, and ribonucleoside UTP; 1% Sarkosyl; 20 U SUPERase-In) preheated to 37°C and incubated for exactly 3 minutes at 37°C. PRO-seq was then performed in duplicate as described (27 (link)). Due to previously determined intrinsic cell type differences in basal transcriptional activity, each of the duplicate LC-2/ad libraries were built using 2 separate run-on reactions (or 20E6 nuclei) as input that were pooled at the first RNA pellet resuspension step. Uniquely indexed libraries were pooled and sequenced on an Illumina NextSeq instrument using 75 bp single-end reads by the BioFrontiers Sequencing Facility at the CU Boulder.

RNA fragments were generated by PCR using specific primers containing the T7 RNA polymerase promoter sequence (CTAATACG ACTCACTATAGGGAGA [T7]) (Supplementary Table 2). Biotinylated transcripts were synthesized in vitro using the MAXIscript Kit (Thermo Fisher Scientific) and the Biotin-11-CTP (PerkinElmer, CT, USA). The synthesized transcripts were then purified with ssDNA/RNA Clean and Concentrator (Zymo Research, CA, USA). After HCT116 cells were lysed with RIPA buffer, whole-cell lysates were prepared by centrifugation at 10,000 g for 10 min at 4° C and used for the biotinylated RNA pull-down assay. Eighty μg of whole-cell lysates were incubated with 2.5 μg of the biotinylated transcripts in TENT buffer (10 mM Tris-HCl buffer, pH 8.0, containing 1 mM EDTA, 250 mM NaCl, and 0.5% Triton X-100) for 30 min at room temperature [5 (link), 11 (link)]. After adding Dynabeads M-280 Streptavidin (Thermo Fisher Scientific), the mixture was incubated for 1 h at room temperature. The bound proteins were pulled down using a magnetic particle separator and analyzed by Western blotting or liquid chromatography-mass spectrometry analysis (nanoLC-MS/MS, CapLC, Q-Tof).