Illumina HiSeq 2000 sequencing; Transcriptional profiling upon knockdown of the RNA-binding protein Unkempt
Transcriptional profiling upon knockdown of the RNA-binding protein Unkempt
HeLa cells were grown in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 micro-g/mL streptomycin at 37  degrees C and 5% CO2.' Lentiviral vectors (pLKO.1) harboring shRNAs targeting human Unkempt were obtained from Sigma (SHCLNG-NM_001080419). To knockdown Unkempt in mouse neurons, we modified the pSilencer1.0 GFP vector (Shen et al., Nature Genetics, 2010) by inserting between the ApaI and EcoRI sites the shRNAs targeting mouse Unkempt mRNA. The targeted sequences (shUnkW: CCCACTCTTCGTGCAGCACAA, shUnk: GGTATCACCTTCGTTACTA) were identified by the Broad Institute RNAi Consortium. SH-SY5Y cells were transfected at 40% confluence using Opti-MEM I Reduced Serum Medium and Lipofectamine 2000 (both Life Technologies) with a pool of siRNA targeting UNK (siGENOME SMART pool, M-022950-01) or a non-targeting siRNA pool (siGENOME Non-Targeting siRNA Pool 1; D-001206-13; both Thermo Scientific Dharmacon) at 25 nM. After 36 hours of transfection, cells were re-transfected with the above siRNA pools and incubated for an additional 24 hours.' Total RNA extracted from triplicates of each control and knockdown cells was DNase-treated and purified using RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. RNA quality was assessed with the 2100 Bioanalyzer (Agilent Technologies).' Sequencing library preparation was carried out using TruSeq RNA Sample Preparation Kit v2 (Illumina).'
Experimental Factor: RNA interference
shUnk Unk knockdown