GSM7612136
Total RNA was extracted using Direct-zol RNA kit (Zymo Research, CA, USA) and rRNA depleted using the NEBNext rRNA depletion kit (human/mouse/rat) (NEB) rRNA-depleted RNA was used for biotinylation via the Click-IT reaction (Click-iT Nascent RNA Capture Kit, ThermoFisher C10365) as per the manufacturer's instructions. First-strand cDNA was synthesised using the captured nascent RNA using the SuperScript VILO cDNA synthesis kit (Invitrogen), followed by purification with 1.8x volume of AMPure XP beads and elution in 20 μL. The second-strand was synthesised using 0.6 mM dATP, 0.6 mM dCTP, 0.6 mM dGTP, and 1.2 mM dUTP in the presence of 2 U of RNase H (Invitrogen) and 20 U of E. coli DNA polymerase I (Invitrogen) in a total volume of 30 μL for 2.5 h at 16°C. cDNA was purified using 1.8x volume AMPure XP beads and eluted in 20 μL of EB. Next, the sequencing libraries were prepared. End-repair was performed in 50 μL of T4 ligase reaction buffer (1×), dNTPs (0.4 nM), T4 DNA polymerase (NEB, 3 U), T4 Polynucleotide Kinase (NEB, 9 U), and Klenow fragment (NEB, 1 U) at 24°C for 30 min in a ThermoMixer at 400 rpm. End-repair reaction was cleaned using 1.8x volume AMPure XP beads and eluted in 15 μL of EB. A-tailing reaction was performed using NEBNext dA-Tailing reaction buffer (NEB, 1×) with Klenow fragment exo- (NEB, 7.5 U) at 37°C for 30 min. A-tailing reaction mixture was mixed with Quick Ligase Buffer (NEB), Quick ligase (NEB, 3,000 U), and 5 nM annealed adaptor (Illumina truncated adaptor) in a volume of 75 μL and incubated at 25°C for 20 min. The adaptor was prepared by annealing the following HPLC-purified oligos: 5′-phosphate/GATCGGAAGAGCACACGTCT-3′ and 5′-ACACTCTTTCCCTACACGACGCTCTTCCGATC∗T-3′ (∗phosphoro-thioate bond). Ligation reaction was stopped by adding 50 mM EDTA, then the DNA was purified with 1.8x volume AMPure XP beads and eluted in 15 μL of EB. Next, 0.5 U of Uracil-DNA glycosylase (ThermoFisher) was added and the mixture was incubated for 15 min at 37°C. PCR was performed in 50 μL reaction mixture with 1 μM primers (TruSeq barcoded primer p5; AATGATACGGCGACCACCGAGATCTACACNNNNNNNNACACTCTTTCCCTACACGACGCTCTTCCGATC∗T; TruSeq barcoded primer p7, CAAGCAGAAGACGGCATACGAGANNNNNNNGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC∗T; NNNNNNNN represents barcode and ∗ a phosphothiorate bond), and 2X Kapa HiFi HotStart Ready mix (Kapa Biosciences). The PCR program was as follows: 98°C for 45 s; 15 cycles of 98°C for 15 s, 63°C for 30 s, and 72°C for 30 s, and a final extension at 72°C for 5 min. PCR products were cleaned with AMPure XP beads, and 200–500 bp fragments were extracted after running on 2% agarose gel. Libraries were purified using QIA-quick Gel Extraction Kit (QIAGEN). Library concentration was determined with KAPA Library Quantification Kit for Illumina Platforms (Kapa Biosystems). Sequencing was performed using Illumina Nextseq 500 (75 bp single-end reads). nascent RNA-seq