GSM4711335: RIC-seq_SARS-CoV-2_rep2; Severe acute respiratory syndrome coronavirus 2; OTHER
After pCp-biotin labelling and proximity ligation, 200 μl of proteinase K buffer (10 mM Tris-HCl pH 7.5, 10 mM EDTA, 0.5% SDS) and 50 μl of proteinase K were applied to beads and sequentially incubated at 37 °C for 60 min and 56 °C for 15 min. The supernatant was isolated by magnet stand and transferred to a new EP tube. The RNA was extracted with 750 μl of TRIzol LS and 220 μl of chloroform following the manufacture's instruction. For fragmentation, 16 μl of total RNA was mixed with 4 μl of 5 × first-strand buffer (250 mM Tris-HCl pH 8.3, 375 mM KCl, 15 mM MgCl2) and incubated at 94 °C for 5 min, and then quickly put on ice. To enrich pCp-biotin marked chimeric RNAs, 20 μl of pre-blocked MyOne Streptavidin C1 beads, 30 μl of NF-H2O and 50 μl of 2 × TWB buffer were applied to each RNA sample and incubated at room temperature for 30 min by rotating. After washing four times with 1× TWB buffer, the beads were resuspended in 100 μl of PK buffer (100 mM NaCl, 10 mM Tris-HCl pH 7.0, 1 mM EDTA, 0.5% SDS) and incubated in Thermomixer at 95 °C for 10 min (1000 rpm). The tube was quickly placed on a magnet stand for 1 min and the supernatant was transferred to a new EP tube. This elution process was repeated two more times. Finally, the enriched RNAs were extracted with acid Phenol: chloroform and precipitated at the final concentration of 300 mM NaCl, with 3 volumes of -20°C ethanol and 1 μl of glycoblue. RNA was pelleted at 13,000 rpm and washed twice with 75% ETOH before resuspending in 10 μl of nuclease-free water. To synthesis first-strand cDNA, 0.5 μl of N6 primer (0.1 μg/μl) was added to the mixture and incubated at 65 °C for 5 min, followed by rapid placement on ice for at least 2 min. Reverse transcription was performed by further addition of 5.5 μl of reverse transcription mix (3 μl of 5 × first-strand buffer, 1 μl of 10 mM dNTPs, 0.5 μl of 100 mM DTT, 0.5 μl of RNase inhibitor, 0.5 μl of Superscript II reverse transcriptase) to the mixture and incubation at 25 °C for 10 min, 42 °C for 40 min and then 70 °C for 15 min. We next created dUTP second strand cDNA by adding the following reaction mixture (10 μl of 5 × second-strand buffer, 0.8 μl of 25 mM dNTPs with 80% of the dTTP replaced by dUTP, 20.5 μl of RNase-free water, 0.2 μl of 5 U/μl RNase H and 2.5 μl of E. coli DNA polymerase I) to the first-strand cDNA mixture, and incubated at 16 °C for 2 h. The double-stranded (ds)DNA products were purified using 1.8 × AMPure beads following the manufacturer's protocol and end-repaired with a mixture of T4 DNA polymerase, Klenow DNA polymerase and T4 polynucleotide kinase. The end-repaired DNA was then treated with Klenow exo- to add an adenine to the 3' ends. For paired-end library construction, adaptors were ligated to dsDNA using quick T4 DNA ligase and incubated at 20 °C for 15 min. Subsequently, the ligated DNA product was purified twice with AMPure beads. The adaptor-ligated cDNA was mixed with 1 μl of 10 μM Illumina PE1.0, 1 μl of 10 μM index primer, 1 μl of 50 mM MgSO4, 2.5 μl of 10 × Pfx buffer, 0.4 μl of 25 mM dNTPs, 0.4 μl of 2.5 U/μl Platinum Pfx DNA polymerase (Thermo Fisher, 11708-013) and 3 μl of USER enzyme (NEB, M5505S). The mixture was incubated at 37 °C for 15 min to allow the USER enzyme to digest the dUTP strand, then at 94 °C for 2 min. PCR was performed with the following programme: 11 to 13 cycles of 94 °C for 15 s, 62 °C for 30 s and 72 °C for 30 s. The 200 to 450 bp of PCR products were purified from agarose gel by Qiaqucik mini Elute Kit. The DNA concentration was measured by Qubit and sequenced by Illumina paired-end sequencing.
GEO Accession
GSM4711335