GSM4677943: THAP1KO_nsRNA_seq_rep3; Mus musculus; RNA-Seq
Four million MEFs were labeled with 0.5 mM 5-ethynyl uridine (EU) for 30 min. Total RNA was extracted using TRIzol (Ambion). The NEBNext rRNA Depletion kit (human/mouse/rat) (New England Biosciences) was used to deplete rRNA from 1 μg of total RNA prior to sample biotinylation through Click-it reactions (Click-iT Nascent RNA Capture Kit, ThermoFisher C10365) as per the manufacturer's specification. First-strand cDNA synthesis of the captured nascent RNA was done using the SuperScript VILO cDNA synthesis kit (Invitrogen), followed by AMPure XP purification (1.8X) and elution in (20 μl), Thereafter, second-strand cDNA synthesis was carried out in a total reaction volume of 30 μl that contained 0.6 mM dNTP, 1.2 mM of dUTP, 2 units of RNase H (Invitrogen) and 20 units of E. coli DNA polymerase I (Invitrogen), for 2.5 hr at 16°C. Double stranded cDNA was cleaned using 1.8X Agencourt AMPure XP beads and eluted in 20 μl of EB that was used for end-repair. End-repair was performed in 50 μl of T4 ligase reaction buffer containing 0.4 mM of dNTPs, 3 units of T4 DNA polymerase (NEB), 9 units of T4 Polynucleotide Kinase (NEB) and 1 unit of Klenow fragment (NEB) at 24°C for 30 min in a ThermoMixer C at 400 rpm. End-repair reaction was cleaned using 1.8X Agencourt AMPure XP beads and eluted in 15 μl of EB that was used for A-tailing reaction in 30 μl of NEBNext dA-Tailing reaction buffer (NEB) with 7.5 units of Klenow fragment exo- (NEB) at 37°C for 30 min. The 30 μl of the A-tailing reaction were mixed with Quick Ligase buffer 2X (NEB), 3,000 units of Quick ligase and 5 nM of annealed adaptor (Illumina truncated adaptor) in a volume of 50 μl and incubated at 25°C for 20 min. The adaptor was prepared by annealing the following HPLC-grade oligos: 5′-Phos/GATCGGAAGAGCACACGTCT-3′and 5′-ACACTCTTTCCCTACACGACGCTCTTCCGATC∗T-3′ (∗phosphorothioate bond). The ligation reaction was terminated by adding 50 mM of EDTA and cleaned with 1.8X Agencourt AMPure XP beads and eluted in 15 μl of EB. Thereafter, samples were treated with 0.5 units of Uracil-DNA glycosylase (Thermofisher) for 15 min at 37°C and used for PCR amplification in a 50 μl reaction volume containing 1 μM of TruSeq barcoded primer p5, AATGATACGGCGACCACCGAGATCTACACNNNNNNNNACACTCTTTCCCTACACGACGCTCTTCCGATC*T, TruSeq barcoded primer p7, CAAGCAGAAGACGGCATACGAGANNNNNNNNGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC*T, (NNNNNNNN represents barcode and * a phosphothiorate bond), and 2X Kapa HiFi HotStart Ready mix (Kapa Biosciences). The temperature settings during the PCR amplification were 45 s at 98°C followed by 15 cycles of 15 s at 98°C, 30 s at 63°C, 30 s at 72°C and a final 5 min extension at 72°C. PCR reactions were cleaned with Agencourt AMPure XP beads (Beckman Coulter), run on a 2% agarose gel and a smear of 200-500bp was cut and gel purified using QIAquick Gel Extraction Kit (QIAGEN). Library concentration was determined with KAPA Library Quantification Kit for Illumina Platforms (Kapa Biosystems). Sequencing was performed on the Illumina Nextseq500 (75 bp single end reads). Nascent RNA-seq
GEO Accession
GSM4677943