GSM7596296: Structure-seq, HEK293T, WT, DMSO, Rep2; Homo sapiens; OTHER
poly(A)-selected RNAs Each library was prepared with 500 ng poly(A)-selected RNAs after 2 rounds of selection (Thermo Fisher Scientific, AM1922). The poly(A) RNAs were fragmented to around 300 nt length at 95°C for 1 min and purified using RNA clean and concentrator (Zymo research, R1016). The fragmented RNA was then subjected to a 5' dephosphorylation reaction at 37°C for 30 min by adding rSAP (NEB, M0371L) and PNK enzyme (NEB, M0201S) in 1× T4 PNK buffer (NEB, B0201S). Next, the 3'adapter was ligated to the dephosphorylated RNA using T4 RNA ligase 2 KQ (NEB, M0373L) at 25°C for 1 h. Excess adapters were digested and removed by adding RecJf (NEB, M0331) and 5' deadenylase (NEB, M0331) for 30 min at 30°C followed by column purification (Zymo research, R1016). The following reverse transcription (RT) was performed in 1 X RT buffer (20 mM Tris, pH 7.5, 4 mM MgCl2, 1 mM DTT, 0.5 mM dNTPs, 150 mM LiCl) with SuperScript III enzyme (Thermo Scientific, 18080093) at 42°C for 40 min. RNA was digested by 2M NaOH and the reaction was neutralized with 1M Tris-HCl (pH 7.5) followed by column purification (Zymo research, R1016). The 5'adapter (for C2C12 libraries) or dU adapter (for HEK293T libraries) was ligated to the cDNA with Quick T4 DNA ligase (NEB, M2200L) and incubated in 37°C overnight. For the C2C12 library, the ligated product was run on the 10% denaturing urea-TBE acrylamide gel (Thermo Scientific, EC68752BOX), and the 100–400 nt region was cut and dissolved in 1X TEN250 buffer (1X TE 7.4, 0.25 M NaCl) at 80°C for 30 min followed by column purification (Zymo research, R1016). For the HEK293T libraries, the ligated product was first purified by column (Zymo research, R1016), then USER II enzyme (NEB, M5509) was added to cleave the product followed by a second column purification (Zymo research, R1016). The purified product was used for PCR cycle test and amplification with different barcoded reverse primer. PCR products with 150–400 bp were then cut and purified with 2% agarose gel and column (Zymo research, D4008).