GSM8411386: SHALiPE-seq, HEK293T, TDP-43 kd, DMSO, Rep2; Homo sapiens; OTHER
Total RNAs were extracted and quantified using RNeasy Plus Mini Kit (Qiagen, 74136) and Nanodrop ND-1000 Spectrophotometer. Poly(A)-selected RNAs were enriched using Poly(A)Purist™ MAG Kit  (Thermo Fisher Scientific, AM1922). 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 was ligated to the cDNA with Quick T4 DNA ligase (NEB, M2200L) and incubated in 37°C overnight. On second day, 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 cut and purified with 2% agarose gel and column (Zymo research, D4008).