GSM9022599: shTDP-43shCTR_input_rep2; Homo sapiens; OTHER
Cells were fixed, permeabilized, blocked, and incubated with anti-DDX6 (Millipore, Cat. No. SAB4200837) and Alexa Fluor 488 (ThermoFisher Scientific). pAG-RTase was added for in situ RT. After proteinase K digestion, nucleic acids were recovered by phenol-chloroform extraction. RNA was digested with RNase H (NEB, Cat. No. M0297S) and RNase A/T1 (Thermo Fisher Scientific, Cat. No. EN0551), followed by biotinylated cDNA enrichment using Dynabeads MyOne Streptavidin C1 (ThermoFisher Scientific, Cat. No. 65001). Next, the 3′ cDNA adapter (5′Phos-8N-AGATCGGAAGAGGTCGTGT-3′SpC3) was ligated by T4 RNA ligase 1 (NEB, Cat. No. M0437M) at 25 °C for 16 hours, and cDNA was recovered with the elution buffer of 95% formamide and 10 mM EDTA (pH 8.0) by boiling at 95°C for 10 min, followed by ethanol precipitation. The library was PCR amplified and gel-purified.